Compounds with reduced ring size for use in diagnosing and treating melanoma, including metastatic melanoma and methods related to same

ABSTRACT

The present invention is directed to novel non-invasive diagnostic tools/compounds to image cancers, especially, melanoma, including metastatic melanoma in vivo. The present compounds exhibit enhanced uptake in cancerous cells and tissue and decreased renal uptake in kidney, evidencing favorable pharmacokinetics of compounds of the present invention. The compounds according to the present invention represent an advance in the diagnosis and treatment of melanoma, including metastatic melanoma using non-invasive molecular imaging techniques. The novel probes of the present invention are also useful for initiating therapy for melanoma as well as monitor patients&#39; response to chemotherapy treatments and other interventions or therapies used in the treatment of melanoma/metastatic melanoma. Compounds according to the present invention may be used as diagnostic tools for a number of conditions and diseases states as well as therapeutic agents for treating such conditions and disease states.

This application is a continuation application of U.S. patentapplication Ser. No. 16/039,858 filed Jul. 19, 2018, which is acontinuation of U.S. patent application Ser. No. 14/622,217 filed Feb.13, 2015, which is a continuation application of U.S. national phasepatent application Ser. No. 13/497,738 filed Apr. 9, 2012 which is a 371application of intetnational patent application number PCT/US2010/058282tiled in the United States Receiving Office on Nov. 30 2010 which claimsthe benefit of priority of United States provisional application serialnumber U.S. 61/283,174, filed Nov. 30, 2009, all of which priorityapplications are incorporated by reference in their entirety herein.

RELATED APPLICATIONS AND GOVERNMENT SUPPORT

The present invention was made with Government support under, grant no.DOD grant W81XWH-09-1-0105 and NIH grant NM-INBRE P20RR016480 from theUnited States DOD/NIH. Consequently, the U.S. government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention is directed to novel non-invasive diagnostictools/compounds to image cancers, especially, melanoma, includingmetastatic melanoma in vivo. The present compounds exhibit enhanceduptake in cancerous cells and tissue and decreased renal uptake inkidney, suggesting favorable pharmacokinetics of compounds of thepresent invention. The compounds according to the present inventionrepresent an advance in the diagnosis and treatment of melanoma,including metastatic melanoma using non-invasive molecular imagingtechniques. The novel probes of the present invention will also beuseful to initiate therapy for melanoma as well as monitor patientsresponse to chemotherapy treatments and other interventions or therapiesused in the treatment of melanoma/metastatic melanoma. Compoundsaccording to the present invention may be used as diagnostic tools for anumber of conditions and diseases states as well as therapeutic agentsfor treating such conditions and disease states.

BACKGROUND OF THE INVENTION

Skin cancer is the most commonly diagnosed cancer in the United States.Melanoma accounts for less than 5% of skin cancer cases but causesgreater than 75% deaths of skin cancer, It was predicted that 68,720 newcases would be diagnosed and 8,650 deaths would occur in 2009 (1). Earlydiagnosis and prompt surgical removal are a patient's best opportunityfor a cure since no curative treatment exists for metastatic melanoma.Despite the clinical use of 2-[¹⁸F]fluoro-2-deoxy-D-glucose ([¹⁸F]FDG)for positron emission tomography (PET) diagnosis and staging ofmelanoma, [¹⁸F]FDG is not melanoma-specific imaging agent and is alsonot effective in imaging small melanoma metastases (<5 mm) and melanomasthat have primary energy sources other than glucose (2-4).Alternatively, melanocortin-1 (MC1) receptor is a distinct moleculartarget due to its over-expression on both human and mouse melanoma cells(5-9). Radiolabeled α-melanocyte stimulating hormone (α-MSH) peptidescan bind the MC1 receptors with nanomolar binding affinities (10-18) andrepresent a class of promising melanoma-specific radiopharmaceuticalsfor melanoma imaging and therapy.

Recently, the inventors have developed a novel class of ¹¹¹In-labeledlactam bridge-cyclized DOTA-conjugated α-MSH peptides for melanomadetection (19, 20). Lactam bridge-cyclization was employed to improvethe stabilities of the α-MSH peptides against the proteolyticdegradations in vivo and enhance the binding affinities of the α-MSHpeptides through stabilizing their secondary structures such as betaturns (21-24). The radiometal chelator DOTA was attached to theN-terminus of the lactam bridge-cyclized α-MSH peptide (12-amino acidsin the peptide ring) for ¹¹¹In radiolabeling. For instance,¹¹¹In-DOTA-GlyGlu-CycMSH(DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp])exhibited high MC1 receptor-mediated tumor uptake (10.40±1.40% ID/g at 2h post-injection) in flank B16/F1 melanoma-bearing C57 mice (19). Bothflank primary and pulmonary metastatic melanoma lesions were clearlyvisualized by small animal SPECT/CT using ¹¹¹In-DOTA-GlyGlu-CycMSH as animaging probe (19, 20), highlighting its potential as an effectiveimaging probe for melanoma detection.

One advantage of the lactam bridge-cycled α-MSH peptide is that thepeptide ring size can be finely modified by either adding or deletingamino acids without sacrificing the binding affinity of the peptide (19,20). The studies on the α-MSH peptide agonists for the MC1 receptorrevealed that the lactam bridge-cyclized α-MSH peptide with a 6-aminoacid peptide ring {Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys(CONH₂)], MT-II}displayed not only higher MC1 receptor binding affinity, but also slowerMC1 receptor dissociation rate than the native α-MSH peptide{Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂} (25, 26).Slow MC1 receptor dissociation rate might contribute to the prolongedbiological activity of MT-II in vitro and in vivo (25). In this study,we conjugated the radiometal chelator DOTA to the N-terminus of theMT-II peptide to generate a novel DOTA-conjugated lactam bridge-cyclizedα-MSH peptide with a 6-amino acid peptide ring (DOTA-Nle-CycMSH_(hex))to examine the effect of peptide ring size on its melanoma targeting andpharmacokinetic properties. The MC1 receptor binding affinity ofDOTA-Nle-CycMSH_(hex) was determined in B16/F1 melanoma cells.DOTA-Nle-CycMSH_(hex) was radiolabeled with ¹¹¹In which is a commercialavailable diagnostic radionuclide with a half-life of 2.8 days. Themelanoma targeting and pharmacokinetic properties and SPECT/CT imagingof ¹¹¹In-labeled DOTA-Nle-CycMSH_(hex) were determined in B16/F1melanoma-bearing C57 mice.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of DOTA-GlyGlu-CycMSH (A),DOTA-Re(Arg¹¹)CCMSH (B) and DOTA-Nle-CycMSH_(hex) (C).

FIG. 2 shows the synthetic scheme of DOTA-Nle-CycMSH_(hex).

FIG. 3 shows the competitive binding curve (A) of DOTA-Nle-CycMSH_(hex)in B16/F1 melanoma cells. The IC₅₀ value of DOTA-Nle-CycMSH_(hex) was1.77 nM. Cellular internalization (B) and efflux (C) of¹¹¹In-DOTA-Nle-CycMSH_(hex) in B16/F1 melanoma cells at 25° C. Totalbound radioactivity (♦), internalized activity (▪) and cell membraneactivity (▴) were presented as counts per minute (cpm).

FIG. 4 shows tumor to kidney uptake ratios of ¹¹¹In-DOTA-GlyGlu-CycMSH,¹¹¹In-DOTA-Nle-CycMSH_(hex) and ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 2, 4 and 24h post-injection. The tumor to kidney uptake ratios of¹¹¹In-DOTA-GlyGlu-CycMSH and ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH were calculatedbased on the results published in the references 19 and 17.

FIG. 5 shows whole-body SPECT/CT images of B16/F1 flank melanoma-bearingC57 mice at 2 (A) and 24 h (B) post-injection of 37.0 MBq of¹¹¹In-DOTA-Nle-CycMSH_(hex). Tumor (T) and kidneys (K) are highlightedwith arrows on the images. HPLC profile (C) of radioactive urine sampleof a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection of¹¹¹In-DOTA-Nle-CycMSH_(hex). ¹¹¹In-DOTA-Nle-CycMSH_(hex) remained intactin the urine 2 h post-injection.

FIG. 6 shows representative compounds according to the present inventionwith certain amino acid and/or peptide tinkers.

FIG. 7 shows representative compounds according to the present inventionwith representative linkers.

FIG. 8 shows the in vitro competitive binding curves ofDOTA-Nle-CycMSH_(hex), DOTA-GGNle-CycMSH_(hex), DOTA-GENle-CycMSH_(hex)and DOTA-NleGE-CycMSH_(hex) in B16/F1 melanoma cells. The IC₅₀ values ofDOTA-Nle-CycMSH_(hex), DOTA-GGNle-CycMSH_(hex), DOTA-GENle-CycMSH_(hex)and DOTA-NleGE-CycMSH_(hex) were 1.8, 2.1, 11.5 and 873.4 nMrespectively. The Data of DOTA-Nle-CycMSH_(hex) was cited from reference19 for comparison.

FIG. 9 shows representative whole-body SPECT/CT images of a B16/F1melanoma-bearing mouse (14 days post cell inoculation) at 2 hpost-injection of 17.0 MBq of ¹¹¹In-DOTA-GGNle-CycMSH_(hex),

FIG. 10 shows the radioactive HPLC profiles of¹¹¹In-DOTA-GGNle-CycMSH_(hex) (injected conjugate) and its metabolitesin urine and tumor at 2 h post-injection.

FIG. 11 shows the kidney (A) and liver (B) uptake values of¹¹¹In-DOTA-Nle-CycMSH_(hex) (▪) and ¹¹¹In-DOTA-GGNle-CycMSH_(hex) (♦).The Data of ¹¹¹In-DOTA-Nle-CycMSH_(hex) was cited from reference 19(second reference set) for comparison.

FIG. 12 shows the tumor/kidney (A) and tumor/liver (B) ratios of¹¹¹In-DOTA-Nle-CycMSH_(hex) (▪) and ¹¹¹In-DOTA-GGNle-CycMSH_(hex) (▪) at2 and 4 h post-injection. The Data of ¹¹¹In-DOTA-Nle-CycMSH_(hex) wascited from reference 19 for comparison.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to compounds according, to the generalstructure:

(Y¹)_(q)-X_(m)-(ABC)_(n)-CycMSH_(hex)

-   Where Y¹ is a chelate group, wherein Y¹ optionally incorporates or    complexes with a radioisotope;-   Each X is independently an amino acid residue (preferably, for    example, a neutral amino acid such as norleucine (Nle), leucine or    isoleucine, more preferably norleucine (Nle), or glycine or alanine,    preferably glycine) which may be optionally acylated (preferably    C₂-C₂₀acylated) at its amino terminal end or an amino acid linker    comprising an alkylene group or an ethylene glycol containing group    according to the chemical structure:

-   ABC is an amino acid linker wherein-   A is absent or is a neutral or negatively charged amino add at    physiological pH which is optionally acylated at its amino terminal    end;-   B is a neutral or negatively charged amino acid at physiological pH    which is optionally acylated (preferably C₂-C₂₀ acylated) at its    amino terminal end;-   C is absent or is a neutral or negatively charged amino acid at    physiological pH;-   m is an integer from 0 to 250, preferably 0 to 5, preferably 0 or 1;-   n is 0 or 1, preferably 1;-   p is an integer from 0 to 20, preferably 0 to 10;-   k is an integer from 0 to 10, preferably 1 or 2;-   Each i is an integer from 0 to 10, preferably 1 or 2;-   Each s is an integer from 0 to 10, preferably 0, 1 or 2, preferably    0;-   q is 0 or 1 (preferably 1), and-   CycMSH_(hex) is a cyclic peptide comprising six amino acids    according to the general structure:

-   Wherein W is a C—H group from an aspartic acid or glutamic acid    residue (preferably an aspartic acid residue), wherein the all    carboxylic acid sidechain of said aspartic acid or glutamic acid and    the alkyleneamine sidechain of lysine are bonded together to form an    amide linkage as indicated;-   X¹ is phenylalanine, tyrosine or tryptophan, preferably    D-phenylalanine;-   Y is arginine or lysine, preferably arginine;-   Z is tryptophan, phenylalanine or tyrosine, preferably tryptophan;-   Z′ is Lys(CONH₂) or Orn(CONH₂), preferably Lys(CONH₂);-   j is 1 or 2 (preferably 1) or-   a pharmaceutically acceptable salt thereof,-   wherein said compound is optionally complexed with at least one    radioisotope preferably a polyvalent cationic radioisotope, even    more preferably selected from the group consisting of ⁸⁶Y, ⁹⁰Y,    ¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, ⁶⁷Cu,    ⁷¹As, ⁷²As, ⁷⁶As, ⁷⁷As, ⁶⁵Zn, ⁴⁸V, ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, ¹⁶⁶Ho,    ¹⁴⁹Pm, ¹⁵³Sm, ²⁰¹Tl, ¹⁸⁸Re, ¹⁸⁶Re and ^(99m)Tc.

In preferred aspects of the invention, the compound incorporates or iscomplexed with a radioisotope as otherwise described herein. In certainaspects of the invention, Y¹ is a radical (i.e., linked to a linker orpeptide as otherwise described herein) of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), CB-TE2A(4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane),NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DTPA(Diethylenetriaminopentaacetic acid), MAG₃ (Mercaptoacetyltriglycine)and 4,5-bis(2-mercaptoacetamido)pentanoic acid and HYNIChydrazinonicotinamide). Other chelating moieties that can complex toradioisotopes are otherwise disclosed herein. In alternative preferredaspects of the invention, CycMSH_(hex) is a cyclic peptide comprisingsix amino acids according to the general structure:

In preferred aspects of the invention, Y¹ is a DOTA radical according tothe chemical structure:

or a pharmaceutically acceptable salt thereof.

In additional preferred aspects of the above-described compounds, n is0, or when n is 1, ABC may be a two or three amino acid unit linker (Aor C may be absent) wherein one, and in certain instances, two or three(preferably, no more than two) of the amino acid units are negativelycharged at physiological pH, e.g. aspartic or glutamic acid, preferablyglutamic acid. In other aspects of the invention, ABC is a three aminoacid unit linker wherein no more than one of the amino acid units isnegatively charged at physiological pH and the other amino acid unitsare neutral at physiological pH. Preferably, the neutral amino acid isnorleucine, leucine, glycine or alanine, preferably norleucine orglycine. X, when present, is preferably a neutral amino acid, preferablynorleucine or leucine, or an alkylene or ethylene glycol containingamino acid linker according to the structure:

as shown above, where p, s, k and i are as otherwise describedhereinabove. It is noted that, compounds according to the presentinvention which contain an ABC amino acid linker (as opposed to thosewithout a linker, i.e., n is 0) and especially a linker having at leastone negatively charged amino acid (e.g., aspartic acid or glutamicacid), often exhibit less renal uptake and consequently enhancedpharmacokinetics (longer half-life in vivo) than do compounds accordingto the present invention which do not contain such linkers. AB linkers(where C is absent) wherein A is glycine or alanine, especially glycineand wherein B is glutamic acid or aspartic acid may also be preferred.In still other embodiments, ABC linkers wherein A is glycine, serine ornorleucine, B is glycine, glutamic acid or aspartic acid and C isglutamic acid (especially when B is glycine) or norleucine (when B isglutamic acid or glycine) may also be preferred. In still otherembodiments, when A and. C are each absent, B is norleucine (Nle),leucine or isoleucine, preferably norleucine (Nle), in particular when mis 0 or X is a PEG linker (e.g. PEG2 linker) as otherwise describedherein.

Alternatively, in certain embodiments, ABC may be preferably GlyGlyGly,GlySerGly, GlyGlyNle, GlyGluNle or NleGlyGlu. Preferred XABC groups (mand n are both 1) include, for example, GlyGlyGlyNle, GlySerGlyNle,GlyAspGlyNle, GlyGluGlyNle and PEG2Nle linkers.

Y¹ is preferably a radical of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), CB-TE2A(4,11-bis(carboxymethyl)-,4,8,11-tetraazabicyclo[6.6.2]hexadecane), NOTA(1,4,7-triazacyclononane-1,4,7-triacetic acid), DTPA(Diethylenetriaminopentaacetic acid), MAG₃ (Mercaptoacetyltriglycine) or4,5-bis(2-mercaptoacetamido)pentanoic acid or HYNIC(hydrazinonicotinamide). More preferably, Y is a radical of DOTA,optionally complexed with a radioisotope as otherwise described herein.

In preferred embodiments, the present invention relates to the abovecompounds, including pharmaceutically acceptable salts, wherein thecompound, especially the Y group, is complexed with a radioisotope(which may be a neutral species or a cationic species, and is preferablya polyvalent cationic species) selected from the group consisting of⁸⁶Y, ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu,⁶⁷Cu, ⁷¹As, ⁷²As, ⁷⁶As, ⁷⁷As, ⁶⁵Zn, ⁴⁸V, ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, ¹⁶⁶Ho,¹⁴⁹Pm, ¹⁵³Sm, ¹⁸⁸Re, ¹⁸⁶Re and ^(99m)Tc.

In further preferred embodiments, Y¹ is a DOTA moiety which may becomplexed with a radioisotope as indicated (this general structure alsocontemplates one or more carbonyl/carboxyl groups in the molecule alsobeing complexed to the radioisotope and is non-limiting) according tothe following:

-   Where Ri is a radioisotope (which may be a neutral species or a    cationic species, and is preferably a polyvalent cationic species)    selected from the group consisting of ⁸⁶Y, ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac,    ²¹²Bi, ²¹³Bi, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, ⁶⁷Cu, ⁷¹As, ⁷⁶As, ⁷⁷As, ⁶⁵Zn,    ⁴⁸V, ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, 164Ho, ¹⁴⁹Pm, ¹⁵³Sm, ²⁰¹Tl, ¹⁸⁸Re, ¹⁸⁶Re    and ^(99m)Tc.

Radioisotopes are selected based on the physical half life, the decaymode (alpha, beta, auger, gamma, X-ray) and the energy of theradioisotope. In diagnostic aspects of the present invention, preferredradioisotopes include, for example, ¹¹¹In, ⁸⁶Y, ⁶⁶Ga, ⁶⁷Ga, ²⁰³Pb, ⁶⁴Cuand ^(99m)Tc.

Where compounds are to be analyzed using positron emission tomography orPET imaging they are labeled with a positron emitting radioisotopes suchas: ⁶⁶Ga, ⁶⁸Ca, ⁶⁴Cu, ⁸⁶Y, or other polyvalent, cationic radiometalsthat decay by positron emission. In alternative embodiments, thecompounds may be analyzed using single photon emission computedtomography or SPECT imaging when labeled with a gamma radiation emittingradioisotope which preferably includes ¹¹¹In, ⁶⁷Ga, ^(99m)Tc and ²⁰³Pbor other gamma emitting radioisotope as disclosed herein.

The present invention relates to compounds and/or compositions which maybe used to prepare imaging/therapeutic agents or as imaging/therapeuticagents (when complexed with a radioisotope) for diagnosing and treatingmelanoma, including metastatic melanoma as otherwise described herein.Compounds according to the present invention which are complexed with anappropriate radioisotope may be used to diagnose the existence and/orextent of melanoma, including metastatic melanoma, monitor therapy as atherapeutic aid of melanoma, including metastatic melanoma, and incertain instances, function as a therapeutic agent (peptide targetedradiation) for the treatment of melanoma, including metastatic melanoma.

The present invention also relates to pharmaceutical compositionscomprising an effective amount of a compound according to the presentinvention which has been complexed with a radioisotope and combined witha carrier, additive or excipient in pharmaceutical dosage form as adiagnostic imaging agent or as a therapeutic agent. Compositionsaccording to the present invention are formulated in pharmaceuticaldosage form for administration preferably by a parenteral, preferably anintravenous route. Compositions according to the present invention mayalso be formulated for administration via a topical route, directly tothe skin. Oral compositions may also be formulated for use in thepresent invention.

In the diagnostic method according to the present invention, a compoundaccording to the present invention is administered to a patient, andevidence of elevated expression of MSH receptors in tissue of saidpatient through standardwell-known nuclear imaging techniques,especially radiation (radionuclide) imaging, including scintigraphicimaging, and especially single photon emission computed tomography(SPECT) and positron emission tomography (PET) in comparison to a normalstandard, is indicative of a disease state (melanoma) and extent ofdisease state (metastasis) in the tissue of the patient. The nuclearimaging techniques useful in the present diagnostic methods are wellknown in the art. In general, elevated levels of radiation emanatingfrom a diagnosed tissue is evidence of elevated MSH receptor activityand indicative of a disease state or condition (melanoma and/ormetastatic melanoma) wherein these receptors are found at elevatedlevels. Methods of diagnosing the existence and/or extent (stage) ofmelanoma, including metastatic melanoma are therefore additional aspectsof the present invention. Thus, a diagnostic method of diagnosing theexistence or absence of melanoma in a patient at risk for melanomacomprises administering to said patient a compound according to thepresent invention; imaging said patient to determine if tissue in saidpatient exhibits elevated expression of MSH receptors; and diagnosingsaid patient as having melanoma, including metastatic melanoma if saidtissue evidences elevated expression of MSH receptors in comparison to astandard.

Methods of monitoring the treatment of melanoma, including metastaticmelanoma in conjunction with traditional or experimental melanomatherapy is an additional aspect of the invention. In this aspect, apatient's response to therapy is monitored using the methods accordingto the present invention. In this method, a patient is monitored beforeand after therapy by administering compound according to the presentinvention and determining (through imaging diagnostics as otherwisedescribed herein) the extent of expression of melanocyte stimulatinghormone receptors in tissues of a patient before therapy and aftertherapy and comparing the expression levels with each other and/or witha standard (predetermined value) to determine the extent of reduction ofcancer tissue which occurred pursuant to the therapeutic intervention.

Methods of treating melanoma represent a further aspect of theinvention, in this aspect, compounds according to the present inventionas described above are administered to a patient known to have melanomaand/or metastatic melanoma in effective amounts in order to reducecancer tissue and otherwise treat the patient's cancer through targetedradiation therapy. The present therapeutic methods may be used alone orin combination with other treatment methods (surgery, chemotherapy,radiation therapy and/or immunotherapy (IL-2 and α-interferon) formelanoma/metastatic melanoma as otherwise disclosed herein. In preferredtherapeutic method aspects of the present invention, compounds accordingto the present invention are labeled with ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹¹⁸⁸Re,212Bi/²¹²Pb, ²¹³Bi, ¹⁴⁹Pm, ¹⁶⁶Ho and ¹⁵⁰Sm and are administered to thepatient (preferably intravenously or topically—i.e, directly onto themelanoma tissue in the skin of the patient) in order to target themalignant melanoma tumor, including metastatic melanoma tissue withradiation therapy.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used to describe the present invention, in theevent that a term is not specifically defined herein, that term isaccorded its commonly understood meaning within the context of its useby those of ordinary skill in the art. It is understood that thedefinitions of the terms which are used to describe the presentinvention are interpreted in a manner consistent with the presentinvention and within the context of a particular term's use indescribing the present invention in one or more embodiments.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a compound”, withincontext, includes a plurality (for example, two or more compounds) ofsuch elements, and so forth. Under no circumstances is the patent beinterpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein.

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human, to whom treatment, includingprophylactic treatment, with the compounds according to the presentinvention is provided. For treatment of those infections, conditions ordisease states which are specific for a specific animal such as a humanpatient, the term patient refers to that specific animal.

The term “compound” is used herein to refer to any specific chemicalcompound disclosed herein. Within its use in context, the term generallyrefers to a single oligopeptide, or an oligopeptide bonded to a DOTAgroup optionally complexed with a radioisotope, but in certain instancesmay also refer to components/portions of such compounds, intermediatesused to synthesize such compounds, stereoisomers and/or optical isomers(including racemic mixtures) of disclosed compounds. The term compoundshall include, where applicable, any and all relevant pharmaceuticallyacceptable salts thereof.

The term “neutral amino acid” is an amino acid which has an unchargedsidechain at physiological pH. Neutral amino acids for use in thepresent invention include, for example, glycine, alanine, valine,leucine, isoleucine, norleucine, methionine, phenylalanine, serine,threonine and tyrosine. Preferred neutral amino acids include glycine,alanine, valine, leucine, isoleucine and norleucine. The term“negatively charged amino acid” is an amino acid which has a negativelycharged sidechain at physiological pH. Preferred negatively chargedamino acids for use in the present invention include glutamic acid andaspartic acid, both of which contain a plurality of carboxylate anions(in contrast to free/protonated carboxylic acids) at physiological pH.

The term “chelate”, “chelator” or “chelating agent” is used to describea moiety (as represented by Y¹ in generic structures) which isfunctionally capable of complexing or “chelating” a radioisotope asotherwise described herein. Each is appropriately chemically linked (viacovalent linkers or directly to Cyclic peptides as otherwise describedherein). Exemplary dictators for use in the present invention, which arewell known in the art, include the following:

-   Polyaminocarboxylates, such as-   EDTA: ethylenediaminetetraacetic acid-   DTPA: diethylenetriaminepentaacetic acid.-   Polyaminocarboxylic Macrocycles, such as:-   DOTA: 1,4,7,10-tetrazacyclodoclecane-1,4,7,10-tetraacetic acid-   TRITA: 1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid-   TETA: triethylenetenamine bridged-cyclam-2a:    1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-1,8-di(methanephosphonic    acid)-   DO3A: 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-   DO2A : 1,4,7,10-tetraazacyclododecane-1,7-bis(acetic acid)-   Other Chelators, such as:-   CB-TE2A    (4,11-bis(carboxymethy)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane)-   NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)-   MAG₃ (Mercaptoacetyltriglycine)-   4,5-bis(2-mercaptoacetamido)pentanoic acid-   HYNIC (hydrazinonicotinamide)

Of the above chelators, DOTA is preferred, as is NOTA and HYNIC. TheNOTA chelator is preferred especially when the radioisotope included isCu or Ga. The HYNIC chelator is preferred, in particular when theradioisotope included is Tc and Re.

Chelates, chelators or chelating agents are generally bi- ormultidentate ligands which generally produce a binding or complexation(complex) of a metal radioisotope as otherwise described herein. Theligand or chelator forms a chelate complex with the substrate. The term,without limitation, is used to describe complexes in which the metal ionis bound to two or more atoms of the chelating agent by whatever means(e.g., coordinate binding or complexation) occurs when a radioisotopeand chelate group complex within each other in compounds according tothe present invention. It is noted here that when a chelator iscomplexed to a radioisotope as used herein, the chelate complexstructure is represented in a generic, nonlimiting sense, such thatbonds which are represented may occur between a radioistope and thechelating agent, as well as additional bonds (such as betweencarbonyl/carboxyl groups) which are not specifically represented, butwhich are understood/determined to be bonded within the context of thechelate complex (to accommodate that different radioisotopes may binddifferently to different chelate groups).

The term “DOTA” is used as an abbreviation for1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, a preferredchelator for use in the present invention, which chemical structure(bonded in compounds according to the present invention) is representedas follows:

Complexed with radioisotopes according to the present invention, DOTAhas the general (note that this general structure also includes thepossibility of carbonyl/carboxyl groups also contributing to the complexdepending on the radioisotope and is non-limiting) structure:

-   Where Ri is a radioisotope as otherwise disclosed herein.

The term“CycMSH_(hex)” or alternatively “cyclic peptide”, “Cycpeptide”“cyclic MSHhex peptide” or refers to cyclic peptides which are boundoptionally through a peptide linker (comprising 1, 2, 3 or 4 amino acidresidues) to DOTA or other chelator according to the present invention.Cyclic peptides according to the present invention may be represented bythe chemical structure:

-   Wherein W is a C—H group from an aspartic acid or glutamic acid    residue (preferably an aspartic acid residue), wherein the all    carboxylic acid sidechain of said aspartic acid or glutamic acid and    the alkyleneamine sidechain of lysine are bonded together to form an    amide linkage as indicated;-   X¹ is phenylalanine, tyrosine or tryptophan, preferably    phenylalanine,-   Y is arginine or lysine, preferably arginine;-   Z is tryptophan, phenylalanine or tyrosine, preferably tryptophan;-   Z′ is Lys(CONH₂) or Orn(CONH₂), preferably Lys(CONH₂);-   j is 1 or 2 (preferably 1) or-   a pharmaceutically acceptable salt thereof,

In preferred aspects of the present invention, X¹ is D-phenylalanine, Yis arginine, Z is tryptophan Z′ is Lys(CONH₂) and is represented by thefollowing chemical structure:

The term “radical” is used to describe a group which is covalentlybonded to another group in compounds according to the present invention.

The term “acylated” is used to describe, an acyl group which may beused, where appropriate, at a terminal amine group of compounds of thepresent invention. The term “acyl” is used throughout the specificationto describe a group at a terminal amine position of an amino acid whichcontains a C₀ to C₂₀ (preferably a C₀ to C₂₀) linear, branched or cyclicalkyl chain. The acyl group at a terminal amine position, results in anamide linkage, which, after administration, may be cleaved. Acyl groupsaccording to the present invention are represented by the structure:

-   where R₄ is H or a C₀ to C₂₀ (preferably, a C₁ to C₂₀) linear,    branched or cyclic alkyl group, phenoxymethyl, aryl, alkoxy,    alkoxyalkyl, aryloxyalkyl, alkoxycarbonyloxy groups (e.g.,    [(isopropoxycarbonyl)oxy]-methoxy), aryloxyalkyl, among others, all    of which groups may be optionally substituted. Preferred acyl groups    are those where R₄ is a C₁ to C₁₀ alkyl group. Acyl groups according    to the present invention also include, for example, those acyl    groups derived from benzoic acid and related acids, 3-chlorobenzoic    acid, succinic, capric and caproic, lauric, myristic, palmitic,    stearic and oleic groups, among numerous others. One of ordinary    skill in the art will recognize the acyl groups which will have    utility in the present invention, either to synthesize the target    pharmaceutical compounds or as prodrug forms of the nucleosides    according to the present invention.

The term “melanoma” is used to describe a malignant tumor of melanocyteswhich are found predominantly in skin but also in the bowel and the eye(see uveal melanoma), even though melanoma can be found in any part ofthe body. Melanoma, is a form of cancer that begins in melanocytes, thecells that make skin pigment, or melanin. It may begin in a mole (skinmelanoma), but can also begin in other pigmented tissues. There areseveral types of melanoma, defined by where they first appear, includingskin and eye melanoma and in rare, instances in the GI tract or lymphnodes

Melanoma is one of the rarer types of skin cancer but causes themajority of skin cancer related deaths. Malignant melanoma is a serioustype of skin cancer. It is due to uncontrolled growth of pigment cells,called melanocytes. Despite many years of intensive laboratory andclinical research, the sole effective cure is surgical resection of theprimary tumor before it achieves a Breslow thickness greater than 1 mm.

Around 160,000 new cases of melanoma are diagnosed worldwide each year.About 48,000 melanoma related deaths occur worldwide per year. Malignantmelanoma accounts for 75 percent of all deaths associated with skincancer. The treatment includes surgical removal of the tumor; adjuvanttreatment; chemo- and immunotherapy, or radiation therapy. The severityof melanoma is often characterized by the Clark level, which are forthin tumors and describe how deeply the cancer has spread into the skin,and the Breslow depth, which refers to the microscopic depth of tumorinvasion.

The following stages are identified in the progression of the melanomadisease state. Melanoma progresses from an early stage (in situ) throughan invasive stage, a high risk melanoma stage, a regional metastaticstage and a distant metastatic stage with varying degrees ofsurvivability, as set forth below.

-   Melanoma stages:-   Stage 0: Melanoma in Situ (Clark Level I), 99.9% Survival-   Stage I/II: Invasive Melanoma, 85-95% Survival

T1a: Less than 1.00 mm primary, w/o Ulceration, Clark Level II-III

T1b: Less than 1.00 mm primary, w/Ulceration or Clark Level IV-V

T2a: 1.00-2.00 mm primary, w/o Ulceration

-   Stage II: High Risk Melanoma, 40-85% Survival

T2b: 1.00-2.00 mm primary, w/Ulceration

T3a: 2.00-4.00 mm primary, w/o Ulceration

T3b: 2.00-4.00 mm primary, w/Ulceration

T4a: 4.00 mm or greater primary w/o Ulceration

T4b: 4.00 mm or greater primary w/Ulceration

-   Stage III: Regional Metastasis, 25-60% Survival

N1: Single Positive Lymph Node

N2: 2-3 Positive Lymph Nodes OR Regional Skin/In-Transit Metastasis

N3: 4 Positive Lymph Nodes OR Lymph Node and Regional Skin/In TransitMetastases

-   Stage IV: Distant Metastasis, 9-15% Survival

M1a: Distant Skin Metastasis, Normal LDH

M1b: Lung Metastasis, Normal LDH

M1c: Other Distant Metastasis OR Any Distant Metastasis with ElevatedLDH

Based Upon AJCC 5-Year Survival With Proper Treatment

Tradition therapy of melanoma involves a number of treatment options.These generally include surgery, chemotherapy, radiation therapy andimmunotherapy (IL-2, other). In the case of surgery, treatment can varyand can include local excision, wide local excision, lymphadenectomy,sentinel lymph node biopsy and skin grafting. In the case ofchemotherapy, a standard chemotherapeutic agent dacarbazine (DTIC) isadministered to the patient in order to treat the cancer, generallythrough cancer cell death. In the case of radiation therapy, radiationis used as a palliative rather than a cure for melanoma. Radiationrelieves bone pain and other symptoms caused by metastases to the bones,brain, and organs such as the liver. Although not curative, radiationtreatment is being investigated for more widespread use in controllingother symptoms of skin cancer. In the case of immunotherapy (biologictreatment), a patient's natural immune system is raised or other immunecompositions (IL-2) are administered to the patient against the cancer.

“Metastatic melanoma” refers to a progressed form of melanoma whereinthe original cancer has metastasized to another area of the skin(regional or distant) or to other non-skin tissue (e.g., lungs, liver,brain, lymph system). Metastatic melanoma describes when melanoma hasspread into surrounding healthy tissue and through the bloodstream, orlymphatic system, to other parts of the body. If melanoma spreads tothese other areas, the cancer cells in the new tumor are still melanomacells but the disease is called metastatic melanoma.

Unlike early stages of melanoma, which can be treated successfully withearly diagnosis, the prognosis for patients diagnosed with metastaticmelanoma is poor, with survival rates of six to nine months. In the past35 years, the FDA has only approved two types of therapies formetastatic melanoma—interleukin 2 (IL-2) and DTIC. The methods oftreatment for metastatic melanoma include radiation, immunotherapy,chemotherapy and palliative surgery. Currently, there are no approvedtherapies that significantly improve survival for patients withmetastatic melanoma

The term “imaging”, “molecular imaging” or “radioimaging is used todescribe methods that use the nuclear properties of matter in diagnosisand therapy, pursuant to the present invention. More specifically, thepresent invention relies on molecular imaging because it produces imagesthat: reflect biological processes that take place at the cellular andsubcellular level.

Molecular imaging is a discipline that unites molecular biology and invivo imaging. It enables the visualisation of the cellular function andthe follow-up of the molecular process in organisms without perturbingthem. The multiple and numerous potentialities of this field areapplicable to the diagnosis and treatment of diseases such as cancer, inthe present invention, in particular, melanoma, including metastaticmelanoma. This technique also contributes to improving the treatment ofthese disorders by optimizing the pre-clinical and clinical tests of newmedication. This approach also has a major economic impact due toearlier and more precise diagnosis.

Molecular imaging differs from traditional imaging in that probeslabeled biomarkers are used to help image particular targets orpathways. Biomarkers interact chemically with their surroundings and inturn alter the image according to molecular changes occurring within thearea of interest. This process is markedly different from previousmethods of imaging which primarily imaged differences in qualities suchas density or water content. This ability to image fine molecularchanges opens up an incredible number of exciting possibilities formedical application, including early detection and treatment of disease,in particular, melanoma and metastatic melanoma according to the presentinvention.

There are a number of different imaging modalities that can be used fornoninvasive molecular imaging, using compounds according to the presentinvention. Each has different strengths and weaknesses and some are moreadept at imaging multiple targets or sites than others. This isimportant in instances where metastatic melanoma is suspected. Themodalities which can be used in the present invention are varied and inthe present invention principally include single photon emissioncomputed tomography (SPECT) and positron emission tomography (PET),discussed below.

The main purpose of SPECT when used in melanoma imaging pursuant to thepresent invention is to measure the distribution of radioisotope in skintissue, in particular, those skin regions and other tissues wheremelanoma, including metastatic melanoma, is suspected. The developmentof computed tomography in the 1970s allowed mapping of the distributionof the radioisotopes in tissue, and led to the technique now calledSPECT.

The imaging agent used in SPECT emits gamma rays, as opposed to thepositron emitters used in PET. There are a number of radioisotopes (suchas ^(99m)Tc, ¹¹¹In, ¹²³I, ²⁰¹Tl, ⁶⁷Ga, ^(99m)Tc, and ²⁰³Pb, among othergamma ray emitters) that can be used in the present invention and imagedwith SPECT technology. In SPECT, where possible, by rotating the gammacamera around the area to be analysed, a three dimensional image of thedistribution of the radiotracer may be obtained by employing filteredback projection or other tomographic techniques. The radioisotopes usedin SPECT have relatively long half lives (a few hours to a few days)making them easy to produce and relatively cheap in comparison to otherradioisotopes. This represents the major advantage of SPECT as animaging technique, since it is significantly cheaper than PET or otherimaging methods such as magnetic resonance imaging (MRI). However, SPECTsometimes lacks exceptional spatial (i.e., where exactly the particleis) or temporal (i.e., did the contrast agent signal happen at aparticular millisecond or not) resolution.

Another imaging technique which finds particular use in the presentinvention is positron emission tomography (PET). In PET, a molecule istagged with a positron emitting isotope. These positrons (β particles)interact with nearby electrons, emitting two 511,000 eV photons,directed 180 degrees apart in opposite directions. These photons arethen detected by the scanner which can estimate the density of positronannihilations in a specific area. When enough interactions andannihilations have occurred, the density of the original molecule may bemeasured in that area. Typical isotopes include ¹¹C, ¹³N, ¹⁵O, ¹⁸F,⁶⁴Cu, ⁶²Cu, ¹²⁴I, ⁷⁶Br, ⁸²Rb and ⁶⁸Ga, among others, including thepreferred ⁶⁶Ga, ⁶⁸Ga, ⁶⁴Cu, ⁸⁶Y. One of the major disadvantages of PETis that most of the radioisotopes must be made with a cyclotron, thusmaking the use of PET, in certain instances prohibitively expensive.Most of these probes also have a half life measured in minutes andhours, thus forcing the cyclotron, in many instances, to be on site.These factors can make PET sometimes prohibitively expensive, except incertain cases, which the present invention addresses in certain aspects.PET imaging does have many advantages though. First and foremost is itssensitivity: a typical PET scanner can detect between 10⁻¹¹ mol/L to10⁻¹² mol/L concentrations.

The term “effective” is used, to describe an amount of a compound,component or composition, which produces an intended effect when usedwithin the context of its use, which may be a diagnostic method, atherapeutic method, a method to monitor the progression of therapy orother method (chemical synthesis) pursuant to the present invention. Inthe case of therapeutic methods, an effective amount for treatingmelanoma, including metastatic melanoma, is that amount which shrinkscancerous tissue (e.g., tumor), produces a remission, prevents furthergrowth of the tumor and/or reduces the likelihood that the cancer in itsearly stages (in situ or invasive) does not progress further tometastatic melanoma.

Noted here is that within the context of the use of the presentinvention, the patient will be receiving a radiation dose, whichprovides guidance to the amount of compound which is consideredeffective when used within the context of its use. A patient undergoinga nuclear medicine procedure will receive a radiation dose. Underpresent international guidelines it is assumed that any radiation dose,however small, presents a risk. The radiation doses delivered to apatient in a nuclear medicine investigation present a very small risk ofside effects, including inducing cancer in the patient. In this respectit is similar to the risk from X-ray investigations except that the doseis delivered internally rather than from an external source such as anX-ray machine.

The radiation dose from a diagnostic nuclear medicine procedure isexpressed as an effective dose with units of sieverts (usually given inmill sieverts, mSv). The effective dose resulting from an investigationis influenced by the amount of radioactivity administered inmegabecquerels (MBq), the physical properties of the radiopharmaceuticalused, its distribution in the body and its rate of clearance from thebody.

Effective doses can range from 6 μSv (0006 mSv) for a 3 MBq chromium-51EDTA measurement of glomerular filtration rate to 37 mSv or more for a150 MBq thallium-201 non-specific tumour imaging procedure. The commonbone scan with 600 MBq of technetium-99m-MDP has an effective dose of 3mSv. Formerly, units of measurement were the Curie (Ci), being 3.7E10Bq, and also 1.0 grams of radium (Ra-226); the rad (radiation absorbeddose), now replaced by the Gray; and the rem (rótgen equivalent man),now replaced with the Sievert. The rad, and rem are essentiallyequivalent for almost all nuclear medicine procedures, and only alpharadiation will produce a higher Rem or Sv value, due to its much higherrelative biological effectiveness (RBE).

The term “coadministration” or “combination therapy” is used to describea therapy in which at least two active compounds (one of which is acompound according to the present invention) in effective amounts areused to treat melanoma, including metastatic melanoma as otherwisedescribed herein at the same time. Although the term coadministrationpreferably includes the administration of two active compounds to thepatient at the same time, it is not necessary that the compounds beadministered to the patient at the same time, although effective amountsof the individual compounds will be present in the patient at the sametime.

-   Compounds according to the present it non may be administered with    one or more compound including a chemotherapeutic agent such as    dacarbazine (DTIC) and/or and immunotherapeutic agent such as IL-2    and/or α-interferon, among other compounds.

The term “treating” “successfully treating” when used in the context oftreating melanoma, including metastatic melanoma, shall includeshrinking a tumor, curing melanoma, including melanoma which hasmetastazied (by causing a remission of the cancer in the patient) orreducing the likelihood or preventing the spread of the melanoma intoother organs. Melanoma, including metastatic melanoma, may be treatedusing compounds according to the present invention alone, or incombination with other methods and/or compounds including surgery,chemotherapy (especially the use of the chemotherapeutic agentdacarbazine or DTIC), radiation therapy (i.e., with agents other thanthe present therapeutic compositions) and immunotherapy (IL-2 and/orα-interferon).

In certain aspects of the invention, where the basic compound and inparticular, the DOTA group, as described above, is complexed with aradioisotope for purposes of being used in the diagnosis or therapy ofmelanoma, including metastatic melanoma, the invention relates tocompounds and their pharmaceutically acceptable salts according to thegeneral chemical structure (note that the radioisotope may be complexedto one or more carbonyl/carboxyl groups of the DOTA moiety as well):

-   Where X, A, B, C, m, n and CycMSH_(hex) are as otherwise described    hereinabove: and the radioisotope (R_(i)) is selected from the group    consisting of ⁸⁶Y, ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, 212Bi, ²¹³Bi, ⁶⁶Ga,    ⁶⁷Ga, ⁶⁸Ga, ⁶⁷Cu, ⁷¹Cu, ⁷¹As, ⁷²As, ⁷⁶As, ⁷⁷As, ⁴⁵Zn, ⁴⁸V, ²⁰³Pb,    ²⁰⁹Pb, ²¹²Pb, ¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁵³Sm, ²⁰¹Tl, ¹⁸⁸Re, ¹⁸⁶Re and ^(99m)Tc,    or a pharmaceutically acceptable salt thereof.

Preferred compounds according to the present invention relate tocompounds according to the structure (note that the radioisotope may becomplexed to one or more carbonyl/carboxyl groups of the DOTA moiety aswell):

Where X, A, B, C, m, n and R_(i) are the same as described above.

Additional preferred compounds according to the present invention may berepresented by the following structure (note that the radioisotope maybe complexed to one or more carbonyl/carboxyl groups of the DOTA moietyas well):

Where Ri is the same as described above, and ABC is a diamino acidlinker (A or C is not present) comprising two amino acids selected fromthe group consisting of neutral amino acids, negatively charged aminoacids or mixtures thereof, and which may be preferably selected from thegroup consisting of GlyGly, LeuGlu, LeuAsp, NleGlu, NleAsp, GlyGlu,GlyAsp, GluGlu, GluAsp, AspGlu and AspAsp, or a pharmaceuticallyacceptable salt thereof. Alternatively, ABC may be preferably GlyGlyGly,GlySerGly, GlyGlyNle, GlyGluNle or NleGlyGlu, as well as GlyGlyGlyNleGlySerGlyNle, GlyAspGlyNle, GlyGluGlyNle and PEG2Nle linkers.

In certain embodiments, the linker X is preferably

-   Wherein p is 2 to 8, k is 1 to 4 (more preferably 1 or 2), s is 0, 1    or 2 (more preferably 0) and i is 1 or 2.

Alternative preferred compounds according to the present invention arerepresented the chemical structure (note that the radioisotope may becomplexed to one or more carbonyl/carboxyl groups of the DOTA moiety aswell):

Wherein X is a neutral amino acid, preferably leucine or norleucine,preferably norleucine (Nle), m is ;1 or 2 and R_(i) is the same asotherwise described above, or a pharmaceutically acceptable saltthereof.

In preferred aspects, R_(i) is selected from the group consisting of¹¹¹In, ⁸⁶Y, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ²⁰³Pb, ⁶⁴Cu and ^(99m)Tc when thecompounds are to be used diagnostically or to monitor therapeuticintervention and R_(i) is selected from the group consisting of ⁹⁰Y,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Bi/²¹²Pb, ²¹³Bi, ¹⁴⁹Pm, ¹⁶⁶Ho and ¹⁵³Sm whencompounds according to the present invention are used in radiationtherapy to treat melanoma, including metastatic melanoma.

The present invention also relates to pharmaceutical compositionscomprising an effective amount of a compound for diagnostic and/ortherapeutic purposes in combination with a pharmaceutically acceptablecarrier, additive or excipient in pharmaceutical dosage form. Fordiagnostic purposes pharmaceutical compositions are formulated generallyin parenteral dosage form, especially for intravenous administration,although oral or topical formulations may be useful in certaininstances. In the case of the use of compounds according to the presentinvention for therapeutic purposes, the compositions are formulatedpreferably in parenteral or topical dosage forms, although orallyadministered dosage forms are also useful.

The compounds of the present invention, may, in accordance with theinvention, be administered in single or divided doses by the oral,parenteral or topical routes. Administration of the active compound mayrange from a single intravenous injection to continuous (intravenousdrip) to several oral administrations per day (for example, Q.I.D.) andmay include oral, topical, parenteral, intramuscular, intravenous,sub-cutaneous, transdermal (which may include a penetration enhancementagent), buccal, sublingual and suppository administration, among otherroutes of administration. Enteric coated oral tablets may also be usedto enhance bioavailability of the compounds from an oral route ofadministration. The most effective dosage form will depend upon thepharmacokinetics of the particular agent chosen as well as the severityof disease in the patient. Administration of compounds according to thepresent invention as sprays, mists, or aerosols for intra-nasal,intra-tracheal or pulmonary administration may also be used. The presentinvention therefore also is directed to pharmaceutical compositionscomprising an effective amount of compound according to the presentinvention, optionally in combination with a pharmaceutically acceptablecarrier, additive or excipient.

The amount of compound used is that amount effective within the contextof the administration, whether that administration is for diagnosticpurposes or therapeutic purposes. A suitable oral dosage for a compoundaccording to the present invention would be in the range of about 0.01mg to 10 g or more per day, preferably about 0.1 mg to about 1 g perday. In parenteral formulations, a suitable dosage unit may contain from0.1 to 250 mg of said compounds, which may be administered from one tofour times per day (for diagnostic purpose, preferably once in a bolusdose), whereas for topical administration, formulations containing 0.01to 1% active ingredient are preferred. It should be understood, however,that the dosage administration from patient to patient will vary and thedosage for any particular patient will depend upon the clinician'sjudgment, who will use as criteria for fixing a proper dosage the sizeand condition of the patient as well as the patient's response to thedrug.

When the compounds of the present invention are to be administered bythe oral route, they may be administered as medicaments in the form ofpharmaceutical preparations which contain them in association with acompatible pharmaceutical carrier, additive or excipient material. Suchcarrier material can be an inert organic or inorganic carrier materialsuitable for oral administration. Examples of such carrier materials arewater, gelatin, talc, starch, magnesium stearate, gum arabic, vegetableoils, polyalkylene-glycols, petroleum jelly and the like.

The pharmaceutical preparations can be prepared in a conventional mannerand finished dosage forms can be solid dosage forms, for example,tablets, dragees, capsules, and the like, or liquid dosage forms, forexample solutions, suspensions, emulsions and the like.

The pharmaceutical preparations may be subjected to conventionalpharmaceutical operations such as sterilization. Further, thepharmaceutical preparations may contain conventional adjuvants such aspreservatives, stabilizers, emulsifiers, flavor-improvers, wettingagents, buffers, salts for varying the osmotic pressure and the like.Solid carrier material which can be used include, for example, starch,lactose, mannitol, methyl cellulose, microcrystalline cellulose, talc,silica, dibasic calcium phosphate, and high molecular weight polymers(such as polyethylene glycol).

For parenteral use, a compound according to the present invention can beadministered in an aqueous or non-aqueous solution, suspension oremulsion in a pharmaceutically acceptable oil or a mixture of liquids,which may contain bacteriostatic agents, antioxidants, preservatives,buffers or other solutes to render the solution isotonic with the blood,thickening agents, suspending agents or other pharmaceuticallyacceptable additives. Additives of this type include, for example,tartrate, citrate and acetate buffers, ethanol, propylene glycol,polyethylene glycol, complex formers (such as EDTA), antioxidants (suchas sodium bisulfite sodium metabisulfite, and ascorbic acid), highmolecular weight polymers (such as liquid polyethylene oxides) forviscosity regulation and polyethylene derivatives of sorbitolanhydrides. Preservatives may also be added if necessary, such asbenzoic acid, methyl or propyl paraben, benzalkonium chloride and otherquaternary ammonium compounds. In certain preferred diagnostic and/ortherapeutic embodiments, compounds according to the present inventionare administered intravenously in sterile saline solution.

The compounds of this invention may also be administered as solutionsfor nasal application and may contain in addition to the compounds ofthis invention suitable buffers, tonicity adjusters, microbialpreservatives, antioxidants and viscosity-increasing agents in anaqueous vehicle. Examples of agents used to increase viscosity arepolyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone,polysorbates or glycerin. Preservatives added may include benzalkoniumchloride, chloro-butanol or phenylethyl alcohol, among numerous others.

Additionally, the compounds provided by the invention can beadministered by suppository.

In certain aspects according to the present invention, where variouscancers are to be treated, the compounds may be co-administered with atleast one other anti-cancer agent such as dacarbazine (DTIC) or an inmunotherapeutic agent such as IL-2 and/or α-interferon. In addition,compounds according to the present invention may be administered priorto, during or after surgery to remove melanoma tissue.

Preparation of compounds according to the present invention proceedsusing standard synthetic chemical techniques which are readily availablein the art. Synthetic methods for obtaining compounds related to thepresent invention may be found in the examples section of the presentspecification. These methods can serve as guides for obtaining compoundsaccording to the present invention. In general, the present compoundsmay be made by condensing an activated DOTA or other chelating group(containing a leaving group or using a coupling agent to facilitate thebinding of the carboxyl group on DOTA or other chelating group to theamine terminal group of the amino acid linker (including, in certaincases, the lysine side chain amine group) or, in the case where thelinker is absent directly to the amine group of the cyclic peptide(CycMSH_(hex)). The radionuclide may be complexed to the chelate (DOTA)group either before or after the activated chelate (DOTA) group iscondensed onto the linker-Cyclic: peptide or directly onto the Cyclicpeptide (linker not present). The linker-cyclic peptide and/or thecyclic peptide with no linker is synthesized using conventional peptidesynthesis (as otherwise described in the examples section or usingmethods readily available in the art using protecting group chemistry)and the various condensation and other reactions, etc. are readilyperformed using methods described herein or otherwise as readily knownin the art. See FIG. 2 hereof for a preferred synthetic approach. Otherapproaches will be readily recognized to those of ordinary skill

Once the compounds are synthesized, they may be formulated inpharmaceutical dosage form using convention pharmaceutical formulationmethods readily available in the art by simply admixing, compounds withchosen carriers, additives and/or excipients, depending upon the dosageform to be used and depending upon the use (diagnostic or therapeutic)of the compositions.

The following examples are provided to assist in describing the presentinvention. The details of these examples and the general description ofthe examples are for description purposes only and should be seen ortaken to limit the scope of the invention in any way.

EXAMPLES—FIRST SET

Materials and Methods

Chemicals and Reagents

Amino acid and resin were purchased from Advanced ChemTech Inc.(Louisville, Ky.) and Novabiochem (San Diego, Calif.). DOTA-tri-t-butylester was purchased from Macrocyclics Inc. (Richardson, Tex.), ¹¹¹InCl₃was purchased from Trace Life Sciences, Inc. (Dallas,Tex.),¹²⁵I-Tyr²-[Nle⁴, D-Phe⁷]-α-MSH }¹²⁵I-(Tyr²)-NDP-MSH} was obtainedfrom PerkinElmer, Inc. (Waltham, Mass.). All other chemicals used inthis study were purchased from. Thermo Fischer Scientific (Waltham,Mass.) and used without further purification. B16/F1 melanoma cells wereobtained from American Type Culture Collection (Manassas, Va.).

Peptide Synthesis

DOTA-Nle-CycMSH_(hex) was synthesized using standardfluorenylmethyloxycarbonyl (Fmoc) chemistry. Briefly, intermediatescaffold of(tBu)₃DOTA-Nle-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde)was synthesized on H₂N-Sieber amide resin by an Advanced ChemTechmultiple-peptide synthesizer (Louisville, Ky.). The protecting group ofDde was removed by 2% hydrazine for peptide cyclization. The protectinggroup of 2-phenylisopropyl was removed and the protected peptide wascleaved from the resin treating with a mixture of 2.5% oftrifluoroacetic acid (TFA) and 5% of triisopropylsilane for 1 h. Afterthe precipitation with ice-cold ether and characterization by liquidchromatography-mass spectroscopy (LC-MS), the protected peptide wasdissolved in H₂O/CH₃CN (30:70) and lyophilized to remove the reagentssuch as TFA and triisopropylsilane. The protected peptide was furthercyclized by coupling the carboxylic group from the Asp with the epsilonamino group from the Lys. The cyclization reaction was achieved by anovernight reaction in dimethylformamide (DMF) usingbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluomphosphate(PyBOP) as a coupling agent in the presence of N,N-diisopropylethylamine(DIEA). After the characterization by LC-MS, the cyclized protectedpeptide was dissolved in H₂O/CH₃CN (30:70) and lyophilized to remove thereagents such as PyBOP and DIEA. The protecting groups were totallyremoved by treating with, a mixture of trifluoroacetic acid (TFA),thioanisole, phenol, water, ethanedithiol and triisopropylsilane(87.5:2.5:2.5:2.5:2.5:2.5) for 4 h at room temperature (25°C.). Thepeptide was precipitated and washed with ice-cold ether four times,purified by reverse phase-high performance liquid chromatography(RP-HPLC) and characterized by LC-MS.

In Vitro Competitive Binding Assay

The IC₅₀ value of DOTA-Nle-CycMSH_(hex) was determined by in vitrocompetitive binding assay according to our previously publishedprocedure (19). B16/F1 cells were harvested and seeded into a 24-wellcell culture plate (5×10⁵ cells/well) and incubated at 37° C. overnight.After being washed twice with binding medium (Dulbecco's ModifiedEagle's Medium with 25 mMN-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid), pH 7.4, 0.2%bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline}, the cells wereincubated at room temperature (25° C.) for 2 h with approximately 60,000cpm of ¹²⁵I-Tyr²-NDP-MSH in the presence of increasing concentrations(10⁻¹² to 10⁻⁵ M) of DOTA-Nle-CycMSH_(hex) in 0.3 mL of binding medium.The reaction medium was aspirated after the incubation. The cells wererinsed twice with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M phosphatebuffered saline (PBS) and lysed in 0.5 mL of 1 N NaOH for 5 minutes. Theactivities associated with cells were measured in a Wallac 1480automated gamma counter (PerkinElmer, N.J.). The IC₅₀ value of thepeptide was calculated using Prism software (GraphPad Software, LaJolla, Calif.).

Peptide Radiolabeling with ¹¹¹In

¹¹¹In-DOTA-Nle-CycMSH_(hex) was prepared in a 0.5 M NH₄OAc-bufferedsolution at pH 4.5 according to our published procedure (19). Briefly,50 μl of ¹¹¹InCl₃ (37-74 MBq in 0.05 M HCl aqueous solution), 10 μL of 1mg/mL DOTA-Nle-CycMSH_(hex) aqueous solution and 400 μL of 0.5 M NH₄OAc(pH 4.5) were added into a reaction vial and incubated at 75° C. for 45min. After the incubation, 10 μL of 0.5% EDTA aqueous solution was addedinto the reaction vial to scavenge potential unbound ¹¹¹In³⁺ ions. Theradiolabeled peptide was purified to single species by Waters RP-HPLC(Milford, Mass.) on a Grace Vydac C-18 reverse phase analytical column(Deerfield, Ill.) using a 20-minute gradient of 18-28% acetonitrile in20 mM HCl aqueous solution with a flow rate of 1.0 mL/min. Purifiedpeptide sample was purged with N₂ gas for 20 minutes to remove theacetonitrile. The pH of final solution was adjusted to 7.4 with 0.1 NNaOH and sterile normal saline for animal studies. In vitro serumstability of ¹¹¹In-DOTA-Nle-CycMSH_(hex) was determined by incubation inmouse serum at 37° C. for 24 h and monitored for degradation by RP-HPLC.

Cellular Internalization and Efflux of ¹¹¹In-DOTA-Nle-CycMSH_(hex)

Cellular internalization and efflux of ¹¹¹In-DOTA-Nle-CycMSH_(hex) wereevaluated in B16/F1 melanoma cells. After being washed twice with thebinding medium, the B16/F1 cells seeded in cell culture plates wereincubated at 25° C. for 20, 40, 60, 90 and 120 min (n=3) in the presenceof approximate 200,000 counts per minute (cpm) of HPLC-purified¹¹¹In-DOTA-Nle-CycMSH_(hex). After incubation, the reaction medium wasaspirated and the cells were rinsed with 2×0.5 mL of ice-cold pH 7.4,0.2% BSA/0.01 M PBS. Cellular internalization of¹¹¹In-DOTA-Nle-CycMSH_(hex) was assessed by washing the cells withacidic buffer [40 mM sodium acetate (pH 4.5) containing 0.9% NaCl and0.2% BSA] to remove the membrane-bound radioactivity. The remaininginternalized radioactivity was obtained by lysing the cells with 0.5 mLof 1 N NaOH for 5 min. Membrane-bound and internalized ¹¹¹In activitieswere counted in a gamma counter. Cellular efflux of¹¹¹In-DOTA-Nle-CycMSH_(hex) was determined by incubating the B16/F1cells with ¹¹¹In-DOTA-Nle-CycMSH_(hex) for 2 h at 25° C., removingnon-specific-bound activity with 2×0.5 mL of ice-cold PBS rinse, andmonitoring radioactivity released into cell culture medium. At timepoints of 20, 40, 60, 90 and 120 min, the radioactivities on the cellsurface and inside the cells were separately collected and counted in agamma counter.

Biodistribution Studies

All the animal studies were conducted in compliance with InstitutionalAnimal Care and Use Committee approval. The mice were housed fiveanimals per cage in sterile micro-isolator cages in a temperature- andhumidity-controlled room with a 12-h light/12-h dark schedule. Thepharmacokinetics of ¹¹¹In-DOTA-Nle-CycMSH_(hex) was determined in B16/F1melanoma-bearing C57 female mice (Harlan, Indianapolis, Ind.). C57 micewere subcutaneously inoculated on the right flank with 1×10⁶ B16/F1cells. The weight of tumors reached approximately 0.2 g 10 days postcell inoculation. Each melanoma-bearing mouse was injected with 0.037MBq of ¹¹¹In-DOTA-Nle-CycMSH_(hex) via the tail vein. Groups of 5 micewere sacrificed at 0.5, 2, 4 and 24 h post-injection, and tumors andorgans of interest were harvested, weighed and counted. Blood valueswere taken as 6.5% of the whole-bodv weight. The tumor uptakespecificity of ¹¹¹In-DOTA-Nle-CycMSH_(hex) was determined byco-injecting 10 μg of unlabeled NDP-MSH, a linear α-MSH peptide analoguewith picomolar affinity for the MC1 receptor present on the melanomacells. To examine whether L-lysine co-injection can reduce the renaluptake or not, a group of 5 mice were injected with a mixture of 12 mgof L-lysine and 0.037 MBq of ¹¹¹In-DOTA-Nle-CycMSH_(hex). The mice weresacrificed at 2 h post-injection. The tumor and organs of interest wereharvested, weighed and counted.

Melanoma Imaging with ¹¹¹In-DOTA-Nle-CycMSH_(hex)

Two B16/F1 melanoma-bearing C57 mice (10 days post the cell inoculation)were injected with 37.0 MBq of ¹¹¹In-DOTA-Nle-CycMSH_(hex) via the tailvein, respectively. The mice were sacrificed for small animal SPECT/CT(Nano-SPECT/CT*, Bioscan) imaging at 2 and 24 h post-injection. The9-min CT imaging was immediately followed by the whole-body SPECTimaging. The SPECT scans of 24 projections were acquired and totalacquisition time was approximately 60 min. Reconstructed data from SPECTand CT were visualized and co-registered using InVivoScope (Bioscan,Washington DC).

Urinary Metabolites of ¹¹¹In-DOTA-Nle-CycMSH_(hex)

The mouse used for the imaging study (2 h post-injection) describedabove was euthanized and the urine was collected for indentifying themetablites. The urinary sample was centrifuged at 16,000 g for 5 minprior to the HPLC analysis. Hie radioactive metabolite in die urine wasanalyzed by injecting aliquots of urine into the HPLC. A 20-minutegradient of 18-28% acetonitrile/20 mM HCl was used for the urineanalysis.

Statistical Analysis

Statistical analysis was performed using the Student's t-test forunpaired data A 95% confidence level was chosen to determine thesignificance between the tumor uptakes of ¹¹¹In-DOTA-Nle-CycMSH_(hex)with or without NDP-MSH co-injection, and between the renal uptakes of¹¹¹In-DOTA-Nle-CycMSH_(hex) with or without L-lysine co-injection in thebiodistribution studies described above. Differences at the 95%confidence level (p<0.05) were considered significant.

Results

To examine the profound effect of the peptide ring size on the melanomaand kidney uptakes of the ¹¹¹In-labeled lactam bridge-cyclized α-MSHpeptide, a novel peptide of DOTA-Nle-CycMSH_(hex) was synthesized andpurified by RP-HPLC. The identity of the peptide was confirmed byelectrospray ionization mass spectrometry (EIMS MW: 1368.5; CalculatedMW; 1368.2). DOTA-Nle-CycMSH_(Hex) displayed greater than 95% puritywith 30% overall synthetic yield. The schematic structures ofDOTA-Nle-CycMSH_(hex) and DOTA-GlyGlu-CycMSH are shown in FIG. 1. FIG. 2illustrates the synthetic scheme of DOTA-Nle-CycMSH_(hex). Thecompetitive binding curve of DOTA-Nle-CycMSH_(hex) is presented in FIG.3A. The IC₅₀ value of DOTA-Nle-CycMSH_(hex) was 1.77 nM in B16/F1 cells.

The peptide was readily labeled with ¹¹¹In in 0.5 M ammonium acetate atpH 4.5 with greater than 95% radiolabeling yield.¹¹¹In-DOTA-Nle-CycMSH_(hex) was completely separated from its excessnon-labeled peptide by RP-HPLC. The retention time of¹¹¹In-DOTA-Nle-CycMSH_(hex) was 10.7 min. ¹¹¹In-DOTA-Nle-CycMSH_(hex)showed greater than 98% radiochemical purity after the HPLCpurification. ¹¹¹In-DOTA-Nle-CycMSH_(hex) was stable in mouse serum at37° C. for 24 h. Only the ¹¹¹In-DOTA-Nle-CycMSH_(hex) was detected byRP-HPLC after 24 h of incubation.

Cellular internalization and efflux of ¹¹¹In-DOTA-Nle-CycMSH_(hex) wereevaluated in B16/F1 cells. FIGS. 3B and 3C illustrate the cellularinternalization and efflux of ¹¹¹In-DOTA-Nle-CycMSH_(hex).¹¹¹In-DOTA-Nle-CycMSH_(hex) exhibited rapid cellular internalization andextended cellular retention. There were 72.9±3.5% and 88.3±0.7% of thecellular uptake of ¹¹¹In-DOTA-Nle-CycMSH_(hex) activity internalized inthe B16/F1 cells at 20 and 120 min post incubation, respectively.Cellular efflux results demonstrated that 89.5±1.9% of internalized¹¹¹In-DOTA-Nle-CycMSH_(hex) activity remained inside the cells 2 h afterincubating cells in culture medium.

The melanoma targeting and pharmacokinetic properties of¹¹¹In-DOTA-Nle-CycMSH_(hex) were determined in B16/F1 melanoma-bearingC57 mice. The biodistribution results of ¹¹¹In-DOTA-Nle-CycMSH_(hex) areshown in Table 1. ¹¹¹In-DOTA-Nle-CycMSH_(hex) exhibited very rapid highmelanoma uptake and prolonged tumor retention in melanoma-bearing mice.At 0.5 h post-injection, ¹¹¹In-DOTA-Nle-CycMSH_(hex) reached its peaktumor uptake value of 24.94±4.58% ID/g. There were 17.01±2.54% ID/g and10.53±1.11% ID/g of the ¹¹¹In-DOTA-Nle-CycMSH_(hex) activity remained inthe tumors at 4 and 24 h post-injection, respectively. In melanomauptake blocking study, the tumor uptake of ¹¹¹In-DOTA-Nle-CycMSH_(hex)with 10 μg of non-radiolabeled NDP-MSH co-injection was only 4.2% of thetumor uptake without NDP-MSH co-injection at 2 h after doseadministration (p<0.05), demonstrating that the tumor uptake wasspecific and MC1 receptor-mediated. Whole-body clearance of¹¹¹In-DOTA-Nle-CycMSH_(hex) was rapid, with approximately 82% of theinjected radioactivity cleared through the urinary system by 2 hpost-injection (Table 1. below). Normal organ uptakes of¹¹¹In-DOTA-Nle-CycMSH_(hex) were low (−1.89% ID/g) except for thekidneys at 2, 4 and 24 h post-injection. High tumor/blood and hightumor/normal organ uptake ratios were achieved as early as 0.5 hpost-injection (Table 1). As the major excretion pathway of¹¹¹In-DOTA-Nle-CycMSH_(hex), the kidney uptake value was 16.20±4.32%ID/g at 0.5 h post-injection and decreased to 9.3±0.91% ID/g at 24 hpost-injection. The tumor to kidney uptake ratios of¹¹¹In-DOTA-Nle-CycMSH_(hex) are presented in FIG. 4. The tumor/kidneyuptake ratios of ¹¹¹In-DOTA-Nle-CycMSH_(hex) were 2.04, 1.70 and 1.13 at2, 4 and 24 h post-injection. Co-injection of NDP-MSH didn't reduce therenal uptake of the ¹¹¹In-DOTA-Nle-CycMSH_(hex) activity at 2 hpost-injection, indicating that the renal uptake was not MC1receptor-mediated. Co-injection of L-lysine significantly (p<0.05)reduced the kidney uptake value by 30% at 2 h post-injection (Table 1).

TABLE 1 Biodistributioa of ¹¹¹In-DOTA-Nle-CycMSH_(hex) in B16/F1melanoma-bearing C57 mice. The data were presented as percent injecteddose/gram or as percent injected dose (Mean ± SD, n = 5) 2 h L- 2 h NDPlysine co- Tissues 0.5 h 2 h 4 h 24 h blockade injection Percentinjected dose/gram (% ID/g) Tumor 24.94 ± 4.58  19.39 ± 1.65  17.01 ±2.54  10.53 ± 1.11   0.81 ± 0.03* 14.48 ± 3.25  Brain 0.21 ± 0.07 0.02 ±0.01 0.06 ± 0.03 0.03 ± 0.01 0.01 ± 0.01 0.04 ± 0.01 Blood 3.33 ± 0.350.11 ± 0.07 0.05 ± 0.02 0.02 ± 0.01 0.07 ± 0.05 0.92 ± 0.48 Heart 1.24 ±0.15 0.16 ± 0.10 0.12 ± 0.03 0.07 ± 0.05 0.06 ± 0.02 0.37 ± 0.02 Lung2.45 ± 0.83 0.32 ± 0.10 0.10 ± 0.05 0.10 ± 0.03 0.30 ± 0.06 0.75 ± 0.21Liver 2.75 ± 0.26 1.46 ± 0.20 1.72 ± 0.07 1.89 ± 0.14 1.46 ± 0.08 1.42 ±0.30 Spleen 1.09 ± 0.33 0.41 ± 0.13 0.47 ± 0.13 0.32 ± 0.08 0.44 ± 0.020.43 ± 0.07 Stomach 3.20 ± 0.98 1.25 ± 0.24 1.49 ± 0.12 1.34 ± 0.42 0.36± 0.14 1.64 ± 0.78 Kidneys 16.20 ± 4.32  9.52 ± 0.44 9.99 ± 1.39 9.31 ±0.91 11.56 ± 0.56   6.66 ± 0.62* Muscle 0.60 ± 0.22 0.15 ± 0.08 0.10 ±0.08 0.03 ± 0.01 0.02 ± 0.01 0.10 ± 0.08 Pancreas 1.18 ± 0.38 0.14 ±0.02 0.16 ± 0.02 0.23 ± 0.08 0.12 ± 0.02 0.21 ± 0.05 Bone 1.34 ± 0.400.18 ± 0.10 0.22 ± 0.15 0.16 ± 0.03 0.05 ± 0.04 0.55 ± 0.14 Skin 4.11 ±0.72 0.66 ± 0.23 0.53 ± 0.05 0.64 ± 0.16 0.29 ± 0.02 1.02 ± 0.09 Percentinjected dose (% ID) Intestines 2.16 ± 0.28 1.40 ± 0.56 3.03 ± 1.06 1.41± 0.86 1.14 ± 0.47 1.85 ± 0.73 Urine 57.00 ± 3.91  82.23 ± 5.83  84.61 ±5.21  87.29 ± 3.60  92.25 ± 1.56  76.79 ± 5.35  Tumor to normal tissueuptake ratio Tumor/Blood 7.49 176.27 340.20 526.50 11.57 15.74Tumor/Kidneys 1.54 2.04 1.70 1.13 0.07 2.17 Tumor/Lung 10.18 60.59170.10 105.30 2.70 19.31 Tumor/Liver 9.07 13.28 9.89 5.57 0.55 10.20Tumor/Muscle 41.57 129.27 170.10 351.00 40.50 144.80 Tumor/Skin 6.0729.38 32.09 16.45 2.79 14.20 *P < 0.05, significance comparison betweenthe tumor uptakes of ¹¹¹In-DOTA-Nle-CycMSH_(hex) with or without NDP-MSHblockade, and between the kidney uptakes of ¹¹¹In-DOTA-Nle-CycMSH_(hex)with or without L-lysine co-injection.

Two B16/F1 melanoma-bearing C57 mice were separately injected with 37.0MBq of ¹¹¹In-DOTA-Nle-CycMSH_(hex) through the tail vein to visualizethe tumors at 2 and 24 h post dose administration. The whole-bodySPECT/CT images are presented in FIGS. 5A and 5B. Flank melanoma tumorswere clearly visualized by SPECT/CT at 2 and 24 h post-injection of¹¹¹In-DOTA-Nle-CycMSH_(hex). Both images showed high tumor to normalorgan uptake ratios except for the kidneys, which was coincident withthe biodistribution results. Urinary metabolite of¹¹¹In-DOTA-Nle-CycMSH_(hex) was analyzed by RP-HPLC 2 h post-injection.FIG. 5C illustrates the urinary HPLC profile of¹¹¹In-DOTA-Nle-CycMSH_(hex). ¹¹¹In-DOTA-Nle-CycMSH_(hex) remained intactin the urine 2 h post-injection.

Discussion

Cyclization strategies using disulfide bridge, lactam bridge and metalcoordination have been successfully employed to cyclize the α-MSHpeptides to enhance the binding affinities and in vivo stabilities ofthe peptides (21-24). Both ¹¹¹In-labeled metal-cyclized and lactambridge-cyclized α-MSH peptides exhibited greater melanoma uptake andlower renal uptake values than those of ¹¹¹In-labeled disulfidebridge-cyclized α-MSH peptide (19, 27). We have reported a novel classof melanoma-specific ¹¹¹In-labeled lactam bridge-cyclized α-MSH peptidesfor both primary and metastatic melanoma imaging (19, 20).¹¹¹In-DOTA-GlyGlu-CycMSH (FIG. 1), with a 12-amino acid peptide ring,exhibited great potential as a melanoma-specific imaging probe indetecting both primary and metastatic melanoma lesions (19, 20).However, the tumor uptake value of ¹¹¹In-DOTA-GlyGlu-CycMSH was 60.15%of the tumor uptake value of ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH, whereas thekidney uptake value of ¹¹¹In-DOTA-GlyGlu-CycMSH was 1.5 times the renaluptake value of ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 2 h post-injection inB16/F1 melanoma-bearing C57 mice (17, 19). The structural differencesbetween ¹¹¹In-DOTA-GlyGlu-CycMSH and ¹¹¹In-DOTA-Re( Arg¹¹)CCMSH (FIG. 1)indicated that smaller size of the peptide ring might contribute to themore favorable melanoma targeting and pharmacokinetic properties of¹¹¹In-DOTA-Re(Arg¹¹)CCMSH since there was a 8-amino acid peptide ring in¹¹¹In-DOTA-Re(Arg¹¹)CCMSH whereas there was a 12-amino acid peptide ringin ¹¹¹In-DOTA-GlyGlu-CycMSH. Moreover, It was reported that the lactambridge-cyclized α-MSH peptide with a 6-amino acid peptide ring{Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys(CONH₂)]} displayed not only higherMC1 receptor binding affinity, but also slower MC1 receptor dissociationrate than the native α-MSH peptide (25). Therefore, we synthesized anovel DOTA-conjugated lactam bridge-cyclized peptide with a 6-amino acidpeptide ring {DOTA-Nle-CycMSH_(hex)} to examine the profound effect ofthe peptide ring size on the tumor and kidney uptakes in this study.

The conjugation of DOTA to the N-terminus of the peptide and foereduction of the peptide ring size did not sacrifice the MC1 receptorbinding affinity of DOTA-Nle-CycMSH_(hex). DOTA-Nle-CycMSH_(hex)exhibited 1.77 nM MC1 receptor binding affinity in B16/F1 melanoma cells(FIG. 3A). ¹¹¹In-DOTA-Nle-CycMSH_(hex) displayed rapid internalizationand prolonged retention in B16/F1 melanoma cells, highlighting itspotential as an effective imaging probe for melanoma detection, as wellas its potential as a therapeutic agent for melanoma treatment whenlabeled with a therapeutic radionuclide. As we anticipated, the strategyof reducing the ring size of the lactam bridge-cyclized α-MSH peptideresulted in improved tumor uptake and prolonged tumor retention.Compared to ¹¹¹In-DOTA-GlyGlu-CycMSH with a 12-amino acid peptide ring.¹¹¹In-DOTA-Nle-CycMSH_(hex) (FIG. 1) only had a 6-amino acid peptidering. The tumor uptake value (19.39±2.72% ID/g) of¹¹¹In-DOTA-Nle-CycMSH_(hex) was 1.86 times the tumor uptake value of¹¹¹In-DOTA-GlyGlu-CycMSH 2 h post-injection in B16/F1 melanoma-bearingC57 mice. ¹¹¹In-DOTA-Nle-CycMSH_(hex) also exhibited prolonged tumorretention than ¹¹¹In-DOTA-GlyGlu-CycMSH. At 24 h post-injection, 54.3%of ¹¹¹In-DOTA-Nle-CycMSH_(hex) activity at 2 h post-injection(10.53±1.11% ID/g) remained in the tumors (Table 1), whereas only 22.8%of the ¹¹¹In-DOTA-GlyGlu-CycMSH radioactivity at 2 h post-injection(2.37±0.28% ID/g) remained inside the tumors. Urinary analysisdemonstrated that the ¹¹¹In-DOTA-Nle-CycMSH_(hex) remained intact 2 hpost-injection (FIG. 5C). It is likely that both high in vivo stabilityof ¹¹¹In-DOTA-Nle-CycMSH_(hex) and low MC1 receptor dissociation rate(15) contributed to the rapid high melanoma uptake (24.94±4.58% ID/g at0.5 h post-injection) and prolonged tumor retention (10.53±1.11% ID/g at24 h post-injection) of ¹¹¹In-DOTA-Nle-CycMSH_(hex) in B16/F1melanoma-bearing C57 mice.

The reduction of the peptide ring size also decreased the non-specifickidney uptake of ¹¹¹In-DOTA-Nle-CycMSH_(hex) compared to¹¹¹In-DOTA-GlyGlu-CycMSH (19) at 2 and 4 h post-injection. The renaluptake values of ¹¹¹In-DOTA-Nle-CycMSH_(hex) were only 72.8% and 82.4%of the renal uptake values of ¹¹¹In-DOTA-GlyGlu-CycMSH at 2 and 4 hpost-injection, respectively. The renal uptake value of¹¹¹In-DOTA-Nle-CycMSH_(hex) was further reduced with L-lysineco-injection by 30% at 2 h post-injection, demonstrating that theelectrostatic interaction between ¹¹¹In-DOTA-Nle-CycMSH_(hex) and thekidney cells played an important role in the renal uptake of¹¹¹In-DOTA-Nle-CycMSH_(hex). The synergistic effects of an increase ofthe tumor uptake and a decrease of the renal uptake dramaticallyimproved the tumor to kidney uptake ratios of¹¹¹In-DOTA-Nle-CycMSH_(hex) at all time points investigated in thisstudy. Improved tumor uptake and decreased kidney uptake resulted insuperior tumor/kidney uptake ratios of ¹¹¹In-DOTA-Nle-CycMSH_(hex) thanthose of ₁₁₁In-DOTA-CycMSH-CycMSH at 2, 4 and 24 h post-injection. Thetumor to kidney uptake ratios of ¹¹¹In-DOTA-Nle-CycMSH_(hex) were 2.55,2.79 and 4.35 times the tumor to kidney uptake ratios of¹¹¹In-DOTA-GlyGlu-CycMSH at 2, 4 and 24 h post-injection, respectively(FIG. 4). ¹¹¹In-DOTA-Nle-CycMSH_(hex) remained intact in the urine 2(FIG. 5C) whereas all of ¹¹¹In-DOTA-CycMSH-CycMSH transformed into twopolar metabolites in the urine 2 h post-injection (19), which mightcontribute to the decreased renal uptake of ¹¹¹In-DOTA-Nle-CycMSH_(hex).

Recently, ^(99m)-labeled lactam bridge-cyclized α-MSH peptides{[Ac-Nle⁴, Asp⁵, D-Phe⁷, Lys¹¹, (pz-^(99m)Tc(CO)₃)]α-MSH₄₋₁₁ and^(99m)(CO)₃-pz-βAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ } have beenreported for melanoma targeting (28, 29).^(99m)Tc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ exhibitedsuperior melanoma uptake (11.31±1.81 % ID/g) to [Ac-Nle⁴, Asp⁵, D-Phe⁷,Lys¹¹ (pz-^(99m)Tc(CO)₃)]α-MSH₄₋₁₁ (4.2435 0.94% ID/g) at 4 hpost-injection in B16/F1 melanoma-bearing C57 mice. However,^(99m)Tc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ displayedhigh accumulation and prolonged retention in both liver (22.86±1.17%ID/g) and kidneys (32.12±1.57% ID/g) at 4 h post-injection, which mightlimit its potential application in metastatic melanoma imaging. In thisstudy, the tumor uptake of ¹¹¹In-DOTA-Nle-CycMSH_(hex) was 1.5 times thetumor uptake of^(99m)Tc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ at 4 hpost-injection, whereas the liver and renal uptake values of¹¹¹In-DOTA-Nle-CycMSH_(hex) were only 7.5% and 31.1% of the^(99m)Tc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ at 4 hpost-injection. Dramatic increase of the tumor uptake and decrease ofthe liver and kidney uptakes of ¹¹¹In-DOTA-Nle-CycMSH_(hex) were likelydue to the structural differences between_(99m)Tc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ and¹¹¹In-DOTA-Nle-CycMSH_(hex).

Currently, metal-cyclized ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH showed the highestmelanoma uptake among all reported ¹¹¹In-labeled linear and cyclic α-MSHpeptides (17). The tumor uptake values of ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH were17.29±2.49, 17.41±5.63 and 8.19±1.63% ID/g at 2, 4 and 24 hpost-injection, respectively (17). Remarkably,¹¹¹In-DOTA-Nle-CycMSH_(hex) exhibited 1.12, 0.98 and 1.29 times thetumor uptake values of ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 2, 4 and 24 hpost-injection, respectively. Meanwhile, ¹¹¹In-DOTA-Nle-CycMSH_(hex)showed slightly higher but similar renal uptake values to¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 2 and 4 h post-injection.¹¹¹In-DOTA-Nle-CycMSH_(hex) exhibited comparable tumor to kidney ratiosas ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 2 and 24 h post-injection despite thatthe tumor to kidney uptake ratio of ¹¹¹In-DOTA-Nle-CycMSH_(hex) was 28%less than that of ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 4 h post-injection. Itwas reported that a single-dose treatment of ²¹²Pb-labeledDOTA-Re(Arg¹¹)CCMSH (200 uCi) resulted in 44% cures in B16/F1melanoma-bearing mice (11). Accordingly, it would be likely that thetreatment of ²¹²Pb-labeled DOTA-Nle-CycMSH_(hex) would yield similarquantitative therapeutic effect for melanoma in the future since¹¹¹In-DOTA-Nle-CycMSH_(hex) displayed comparable tumor to kidney ratiosas ¹¹¹In-DOTA-Re(Arg¹¹)CCMSH at 2 and 24 h post-injection.

Conclusion

The ring size of the ¹¹¹In-labeled lactam bridge-cyclized α-MSH peptideexhibited a profound effect on its melanoma targeting andpharmacokinetic properties. The reduction of the peptide ring sizedramatically increased the melanoma uptake and decreased the renaluptake of ¹¹¹In-DOTA-Nle-CycMSH_(hex), providing a new insight into thedesign of novel radiolabeled lactam bridge-cyclized α-MSH peptide formelanoma imaging and treatment.

FURTHER EXAMPLES

Materials and Methods

Chemicals and Reagents

Amino acids and resin were purchased from Advanced ChemTech Inc,(Louisville, Ky.) and Novabiochem (San Diego, Calif.). DOTA-tri-t-butylester was purchased from Macrocyclics Inc. (Richardson, Tex.) forpeptide synthesis, ¹²⁵I-Tyr²-[Nle⁴, D-Phe⁷]-α-MSH {¹²⁵I-(Tyr²)-NDP-MSH}was obtained from PerkinElmer, Inc. (Waltham, Mass.) for in vitroreceptor binding assay. ¹¹¹InCl₃ was purchased from Trace Life Sciences,Inc. (Dallas, Tex.) for radiolabeling. All other chemicals used in thisstudy were purchased from Thermo Fischer Scientific (Waltham, Mass.) andused without further purification. B16/F1 murine melanoma, cells wereobtained from American Type Culture Collection (Manassas, Va.).

Peptide Synthesis

New DOTA-GGNle-CycMSH_(hex), DOTA-GENle-CycMSH_(hex) andDOTA-NleGE-CycMSH_(hex) peptides were synthesized using standardfluorenylmethyloxycarbonyl (Fmoc) chemistry according to the inventors'published procedure (19, second set of references) with modifications.Briefly, linear peptide backbones of(tBu)₃DOTA-Gly-Gly-Nle-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde),(tBu)DOTA-Gly-Glu(OtBu)-Nle-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde)and(tBu)DOTA-Nle-Gly-Glu(OtBu)-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde)were synthesized on Sieber Amide resin by an Advanced ChemTechmultiple-peptide synthesizer (Louisville, Ky.). Seventy micromoles ofresin, 210 μmol of each Fmoc-protected amino acid and 210 μmol of(tBu)₃DOTA were used for the synthesis. The protecting group of Dde wasremoved by 2% hydrazine for peptide cyclization. The protecting group of2-phenylisopropyl was removed and the protected peptide, was cleavedfrom the resin treating with a mixture of 2.5% of trifluoreacetic acid(TFA) and 5% of triisopropylsilane. After the precipitation withice-cold ether and characterization by liquid chromatography-massspectroscopy (LC-MS), each protected peptide was dissolved in H₂O/CH₃CN(50:50) and lyophilized to remove the reagents. Then, each protectedpeptide was further cyclized by coupling the carboxylic group from theAsp with the epsilon amino group from the Lys. The cyclization reactionwas achieved by an overnight reaction in dimethylformamide (DMF) usingbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-bexafluorophospbate(PyBOP) as a coupling agent in the presence ofN,N-diisoprorpylethylamine (DIEA). After the characterization by LC-MS,each cyclized protected peptide was dissolved in H₂O/CH₃CN (50:50) andlyophilized to remove the reagents. The protecting groups were totallyremoved by treating with a mixture of trifluoroacetic acid (TFA),thioanisole, phenol, water, ethanedithiol and triisopropylsilane(87.5:2.5:2.5:2.5:2.5:2.5) for 2 h at room temperature (25° C.). Eachpeptide was precipitated and washed with ice-cold ether four times,purified by reverse phase-high performance liquid chromatography(RP-HPLC) and characterized by LC-MS.

In Vitro Receptor Binding Assay

The receptor binding affinities (IC₅₀ values) of DOTA-GGNle-CycMSH_(hex)DOTA-GENle-CycMSH_(hex) and DOTA-NleGE-CycMSH_(hex) were determined byin vitro competitive binding assay according to the published procedure(19) with modifications. B16/F1 cells in 24-well cell culture plates(5×10⁵ cells/well) were incubated at room temperature (25° C.) for 2 hwith approximately 60,000 cpm of ¹²⁵I-Tyr²-NDP-MSH in the presence of10⁻¹² to 10⁻⁵ M of each peptide in 0.3 mL of binding medium {Dulbecco'sModified Eagle's Medium with 25 mMN-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid), pH 7.4, 0.2%bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline}. The medium wasaspirated after the incubation. The cells were rinsed twice with 0.5 mL,of ice-cold pH 7.4, 0.2% BSA/0.01 M phosphate buffered saline (PBS) andlysed in 0.5 mL of 1 N NaOH for 5 minutes. The activities associatedwith cells were measured;in a Wallac 1480 automated gamma counter(PerkinElmer, Waltham, Mass.). The IC₅₀ value of each peptide wascalculated using Prism software (GraphPad Software, La Jolla, Calif.).

Peptide Radiolabeling with ¹¹¹In

Since DOTA-NleGE-CycMSH_(hex) exhibited at least 78-fold lower receptorbinding affinity than DOTA-GGNle-CycMSH_(hex) andDOTA-GENle-CycMSH_(hex), we only further evaluatedDOTA-GGNle-CycMSH_(hex) and DOTA-GENle-CycMSH_(hex).¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex) wereprepared in a 0.5 M NH₄OAc-buffered solution at pH 4.5 according to ourpublished procedure (19). Briefly, 50 μL of ¹¹¹InCl₃ (37-74 MBq in 0.05M HCl aqueous solution), 10 μL of 1 mg/mL peptide aqueous solution and400 μL of 0.5 M NH₄OAc (pH 4.5) were added into a reaction vial andincubated at 75° C. for 45 mins. After the incubation, 10 μL, of 0.5%EDTA (ethylenediaminetetraacetic acid) aqueous solution was added intothe reaction vial to scavenge potential unbound ¹¹¹In³⁺ ions. Theradiolabeled complexes were purified to single species by Waters RP-HPLC(Milford, Mass.) on a Grace Vydac C-18 reverse phase analytical column(Deerfield, Ill.) using the following gradient at a 1 mL/min flowrate.The mobile phase consisted of solvent A (20 mM HCl aqueous solution) andsolvent B (100% CH₃CN). The gradient was initiated and kept at 82:18 A/Bfor 3 mins followed by a linear gradient of 82:18 A/B to 72:28 A/B over20 mins. Then, the gradient was changed from 72:28 A/B to 10:90 A/B over3 mins followed by an additional 5 mins at 10:90 A/B. Thereafter, thegradient was changed from 10:90 A/B to 82:18 A/B over 3 mins. Eachpurified peptide sample was purged with gas for 20 mins to remove theacetonitrile. The pH of the final solution was adjusted to 7.4 with 0.1N NaOH and sterile saline for animal studies. In vitro serum stabilityof ¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex) weredetermined by incubation in mouse serum at 37° C. for 24 h and monitoredfor degradation by RP-HPLC.

Biodistribution Studies

All animal studies were conducted in compliance with InstitutionalAnimal Care and Use Committee approval. The pharmacokinetics¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex) weredetermined in B16/F1 melanoma-bearing C57 female mice (Harlan,Indianapolis, Ind.). The C57 mice were subcutaneously inoculated with1×10⁶ B16/F1 cells on the right flank for each mouse to generate B16/F1melanomas. Ten days post inoculation, the tumor weights reachedapproximately 0.2 g. Each melanoma-bearing mouse was injected with 0.037MBq of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) or ¹¹¹In-DOTA-GENle-CycMSH_(hex)via the tail vein. Groups of 5 mice were sacrificed at 0.5, 2, 4 and 24h post-injection, and tumors and organs of interest were harvested,weighed and counted. Blood values were taken as 6.5% of the whole-bodyweight. The specificities of the tumor uptake of¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex) weredetermined by co-injecting 10 μg (6.07 nmol) of unlabeled NDP-MSH whichis a linear α-MSH peptide analogue with picomolar MC1 receptor bindingaffinity.

Melanoma Imaging

Since ¹¹¹In-DOTA-GGNle-CycMSH_(hex) displayed more favorable tumortargeting and pharmacokinetic properties than¹¹¹In-DOTA-GENle-CycMSH_(hex), we only further evaluated the melanomaimaging property of ¹¹¹In-DOTA-GGNle-CycMSH_(hex). One B16/F1melanoma-bearing C57 mouse (10 days post the cell inoculation) wasinjected with 37.0 MBq of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) via the tailvein. The mouse was sacrificed for small animal SPECT/CT(Nano-SPECT/CT®, Bioscan) imaging at 2 h post-injection. The CT imagingwas immediately followed by the whole-body SPECT imaging. The SPECTscans of 24 projections were acquired. Reconstructed SPECT and CT datawere visualized and co-registered using InVivoScope (Bioscan,Washington, DC).

Metabolites of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) in Melanoma and Urine

Both melanoma and urine were collected from the mouse used for SPECT/CTimaging to analyze the metabolites of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) inmelanoma and urine. The tumor was homogenized by a VWR homogenizer for 5mins. Equal volume of ethanol was added into the tumor sample. The tumorsample was vortexed and then centrifuged at 16,000 g for 5 mins. Thesupernatant was transferred into a glass test tube and purged with N₂gas for 20 mins to remove the ethanol. Aliquots of the supernatant wereinjected into HPLC. The urinary sample was directly centrifuged at16,000 g for 5 mins prior to the HPLC analysis. Thereafter, aliquots ofthe urine were injected into HPLC. The HPLC gradient described above wasused for the analyses of metabolites.

Statistical Analysis

Statistical analysis was performed using the Student's t-test forunpaired data. A 95% confidence level was chosen to determine thesignificant difference in tumor and renal uptakes between¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex), as wellas the significant difference in tumor and renal uptakes between¹¹¹In-DOTA-GGNle-CycMSH_(hex) or ¹¹¹In-DOTA-GENle-CycMSH_(hex)with/without NDP-MSH co-injection. The differences at the 95% confidencelevel (p<0.05) were considered significant.

Results

Three novel α-MSH peptides, DOTA-GGNle-CycMSH_(hex),DOTA-GENle-CycMSH_(hex) and DOTA-NleGE-CycMSH_(hex) were synthesized andpurified by HPLC. All three peptides displayed greater than 95% purityafter HPLC purification. The schematic structures of the peptides areshown in FIG. 6. The identities of the peptides were confirmed byelectrospray ionization mass spectrometry. The calculated and foundmolecular weights of the peptides are presented in Table 1A. Thereceptor binding, affinities of the peptides were determined in B16/F1melanoma cells. The IC₅₀ values of DOTA-GGNle-CycMSH_(hex),DOTA-GENle-CycMSH_(hex) and DOTA-NleGE-CycMSH_(hex) were 2.1, 11.5 and873.4 nM in B16/F1 cells, respectively (Table 1 and FIG. 8),

Only DOTA-GGNle-CycMSH_(hex) and DOTA-GENle-CycMSH_(hex) displayed lownanomolar MC1 receptor binding affinities. Hence, we only furtherevaluated DOTA-GGNle-CycMSH_(hex) and DOTA-GENle-CycMSH_(hex).DOTA-GGNle-CycMSH_(hex) and DOTA-GENle-CycMSH_(hex) were readily labeledwith ¹¹¹In in 0.5 M ammonium acetate solution at pH 4.5 with greaterthan 95% radiolabeling yield. Each ¹¹¹In-labeled peptide was completelyseparated from its excess non-labeled peptide by RP-HPLC. The retentiontimes of the peptides and their ¹¹¹In-labeled conjugates are showed inTable 1A. The retention times of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) and¹¹¹In-DOTA-GENle-CycMSH_(hex) were 17.7 and 21.7 min, respectively.¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex) showedgreater than 98% radiochemical purities after HPLC purification, andwere stable in mouse serum at 37° C. for 24 h. Only intact ¹¹¹In-labeledconjugates were detected by RP-HPLC after 24 h of incubation in, mouseserum.

The inventors further evaluated the melanoma targeting andpharmacokinetic properties of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) and¹¹¹In-DOTA-GENle-CycMSH_(hex) in B16/F1 melanoma-bearing C57 mice. Thebiodistribution results of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) and¹¹¹In-DOTA-GENle-CycMSH_(hex) are shown in Table 2A.¹¹¹In-DOTA-GGNle-CycMSH_(hex) exhibited rapid high melanoma uptake andprolonged tumor retention. The tumor uptake value of¹¹¹In-DOTA-GGNle-CycMSH_(hex) was 18.39±2.22% ID/g at 0.5 hpost-injection. The tumor uptake reached its peak value of 19.05±5.04%ID/g at 2 h post-injection. ¹¹¹In-DOTA-GGNle-CycMSH_(hex) displayedsimilar high tumor uptake (18.6±3.56% ID/g) at 4 h post-injection. Evenat 24 h post-injection, there was 6.77±0.84% ID/g of¹¹¹In-DOTA-GGNle-CycMSH_(hex) activity remained in the tumor.Approximately 98% of the tumor uptake of ¹¹¹In-DOTA-GGNle-CycMSH_(hex)was blocked by 10 μg 7 nmol) of non-radiolabeled NDP-MSH (p<0.05),demonstrating that the tumor uptake was specific and MC1receptor-mediated. Whole-body clearance of ¹¹¹In-DOTA-GGNle-CycMSH_(hex)was rapid, with approximately 88.4% of the injected radioactivitycleared through the urinary system by 2 h post-injection (Table 2A).Normal organ uptakes of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) were low (<1.31%ID/g) except for the kidneys 2, 4 and 24 h post-injection. The liveruptake of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) was less than 0.61% ID/g at 2 hpost-injection (Table 2A). The kidney uptake value was 15.19±2.75% ID/gat 0.5 h post-injection, and decreased to 6.84±0.92% ID/g at 2 hpost-injection (Table 2A). Co-injection of NDP-MSH didn't significantlyreduce the renal uptake of the ¹¹¹In-DOTA-GGNle-CycMSH_(hex) activity at2 h post-injection, indicating that the renal uptake was not MC1receptor-mediated. High tumor uptake and prolonged tumor retentioncoupled with rapid whole-body clearance resulted in high tumor/blood andhigh tumor/normal organ uptake ratios that were achieved as early as 0.5h post-injection (Table 2A). The tumor/liver uptake ratios of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) were 33.42 and 31.0 at 2 and 4 hpost-injection, whereas the tumor/kidney uptake ratios of¹¹¹In-DOTA-GGNle-CycMSH_(hex) were 2.79 and 2.73 at 2 and 4 hpost-injection.

¹¹¹In-DOT-GENle-CycMSH_(hex) showed lower tumor uptake values than¹¹¹In-DOTA-GGNle-CycMSH_(hex) at 0.5, 2 and 4 h post-injection. Thetumor uptake values of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) were 2, 2.5 and 3times the tumor uptake values of ¹¹¹In-DOTA-GENle-CycMSH_(hex) at 0.5, 2and 4 h post-injection, respectively (Table 2A). Co-injection ofnon-radioactive NDP-MSH blocked 95.6% of the tumor uptake at 2 hpost-injection (p<0.0.5), indicating that the tumor uptake of¹¹¹In-DOTA-GENle-CycMSH_(hex) was MC1 receptor-specific. Despite thesimilar renal uptake of ¹¹¹In-DOTA-GENle-CycMSH_(hex) as¹¹¹In-DOTA-GGNle-CycMSH_(hex) at 2, 4 and 24 h post-injection,¹¹¹In-DOTA-GENle-CycMSH_(hex) showed 40% lower renal uptake than¹¹¹In-DOTA-GGNle-CycMSH_(hex) at 0.5 h post-injection (p<0.05). Thekidney uptake of ¹¹¹In-DOTA-GENle-CycMSH_(hex) was as low as 9.06±2.20%ID/g at 0.5 h post-injection and decreased to 5.54±0.63% ID/g at 2 hpost-injection.

We further evaluated the melanoma imaging properties of¹¹¹In-DOTA-GGNle-CycMSH_(hex) since ¹¹¹In-DOTA-GGNle-CycMSH_(hex) showedmore favorable biodistribution properties than¹¹¹In-DOTA-GENle-CycMSH_(hex). The whole-body SPECT/CT images arepresented in FIG. 9. Flank melanoma tumors were clearly visualized bySPECT/CT using ¹¹¹In-DOTA-GGNle-CycMSH_(hex) as an imaging probe. Thewhole-body images showed high tumor to normal organ uptake ratios exceptfor the kidneys, which was consistent with the biodistribution results.Melanoma and urinary metabolites of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) wereanalyzed by RP-HPLC 2 h post-injection. FIG. 10 illustrates both theHPLC profiles of melanoma and urine samples.¹¹¹In-DOTA-GGNle-CycMSH_(hex) remained intact in the both tumor andurine 2 h post-injection (FIG. 10).

Discussion

The present inventors have been interested in developing lactambridge-cyclized α-MSH peptides to target the MC1 receptors for melanomadetection (15-19). Unique lactam bridge-cyclization makes the cyclicα-MSH peptides resistant to proteolytic degradations in vivo, as well asprovides the flexibility for fine structural modification (15, 17, 19).Recently, we have identified ¹¹¹In-DOTA-Nle-CycMSH_(hex) with a 6-aminoacid ring targeting the MC1 receptors for melanoma imaging (19). Amongthese reported lactam bridge-cyclized α-MSH peptides (15, 17, 19),¹¹¹In-DOTA-Nle-CycMSH_(hex) displayed the highest melanoma uptake values(24.94±4.58% ID/g at 0.5 h post-injection and 19.39±1.65% ID/g at 2 hpost-injection) in B16/F1 melanoma-bearing mice (19). The reduction ofthe ring size improved the tumor uptake and reduced the renal uptake of¹¹¹In-DOTA-Nle-CycMSH_(hex), providing a new insight into the design ofnovel lactam bridge-cyclized α-MSH peptides for melanoma targeting.

Hydrocarbon, amino acid and PEG linkers have been used to optimize thereceptor binding affinities, as well as modifying the pharmacokineticproperties of radiolabeled bombesin (21-25), RGD (26-29) and α-MSHpeptides (15, 16). For instance, Volkert and colleagues reported thatthe hydrocarbon linkers ranging from 5-carbon to 8-carbon between theDOTA and bombesin peptide resulted in 0.6-1.7 nM receptor bindingaffinities for the DOTA-conjugated bombesin peptides. Ether shorter orlonger hydrocarbon linkers dramatically reduce the receptor bindingaffinity by 100-fold (21). Rogers and colleagues reported the profoundeffects of amino acid linkers (-GlyGlyGly-, -GlySerGly-, -GlySerSer- and-GlyGluGly-) between the DOTA and bombesin peptide on tumor and normalorgan uptakes of the radiolabeled peptides (25). ⁶⁴Cu-labeledDOTA-conjugated bombesin peptide with the -GlyGlyGly-linker displayedthe higher PC-3 tumor uptake, whereas the -GlySerGly-linker resulted inlower renal uptake (25). Recently, Liu and colleagues reported theimprovement in tumor uptakes and pharmacokinetics of ⁶⁴Cu- and^(96m)Tc-labeled cyclic RGD peptides using the -GlyGlyGly- and PEG₄linkers (26-29). We also demonstrated that the introduction of anegatively-charged -GlyGlu-linker enhanced the melanoma uptake andreduced the renal uptake of ¹¹¹In-DOTA-GlyGlu-CycMSH compared to¹¹¹In-DOTA-CycMSH (15). Hence, we evaluated the effects of -GlyGly- and-GlyGlu-linkers on melanoma targeting and pharmacokinetic properties of¹¹¹In-DOTA-[X]-CycMSH_(hex) peptide constructs in this study.

DOTA-Nle-CycMSH_(hex) displayed 1.8 nM MC1 receptor binding affinity inB16/F1 melanoma cells in our previous report (19). The MC1 receptorbinding sequence of His-dPhe-Arg-Trp was directly cyclized by an Asp-Lyslactam bridge to generate the CycMSH_(hex) moiety. The radiometalchelator DOTA was conjugated to the CycMSH_(hex) moiety via a Nle toform DOTA-Nle-CycMSH_(hex) peptide. Based on the unique structure ofDOTA-Nle-CycMSH_(hex), we initially introduced the amino acid linker(-GlyGlu-) between the DOTA and Nle or between the Nle and CycMSH_(hex)moiety to determine which position was suitable for an amino acidlinker. We found that the moiety of Nle-CycMSH_(hex) was critical formaintaining the low nanomolar MC1 receptor binding affinity of thepeptide. The introduction of the -GlyGlu-linker between the Nle andCycMSH_(hex) moiety dramatically reduced the MC1 receptor bindingaffinity to 873.4 nM, whereas the introduction of the -GlyGlu-linkerbetween the DOTA and Nle only decreased the MC1 receptor bindingaffinity to 11.5 nM. Interestingly, the -GlyGly-linker between the DOTAand Nle maintained the MC1 receptor binding affinity as 2.1 nM, furtherindicating the moiety of Nle-CycMSH_(hex) played a crucial role inmaintaining the low nanomolar MC1 receptor binding affinity of thepeptide. The difference in MC1 receptor binding affinity betweenDOTA-GGNle-CycMSH_(hex) and DOTA-GENle-CycMSH_(hex) (2.1 nM vs. 11.5 nM)was also observed in the melanoma uptakes of¹¹¹In-DOTA-GGNle-CycMSH_(hex) and ¹¹¹In-DOTA-GENle-CycMSH_(hex) inB16/F1 melanoma-bearing C57 mice. The tumor uptake values of¹¹¹In-DOTA-GGNle-CycMSH_(hex) were 2, 2.5 and 3 times the tumor uptakevalues of ¹¹¹In-DOTA-GENle-CycMSH_(hex) at 0.5, 2 and 4 hpost-injection, respectively (Table 2A). In our previous report, theintroduction of a negatively-charged -GlyGlu-linker resulted in 44%lower renal uptake of ¹¹¹In-DOTA-GlyGlu-CycMSH at 4 h post-injectioncompared to ¹¹¹In-DOTA-CycMSH (15). In this study,¹¹¹In-DOTA-GENle-CycMSH_(hex) showed 40% lower renal uptake (p<0.05)than ¹¹¹In-DOTA-GGNle-CycMSH_(hex) at 0.5 h post-injection (Table 2A).

At the present time, the lactam bridge-cyclized¹¹¹In-DOTA-Nle-CycMSH_(hex) and the metal-cyclized¹¹¹In-DOTA-Re(Arg¹¹)CCMSH displayed the highest comparable melanomauptakes among all reported ¹¹¹In-labeled linear and cyclic α-MSHpeptides (13, 19). The melanoma uptake values were 17.29±2.49 and17.41±5.63% ID/g at 2 and 4 h post-injection for¹¹¹In-DOTA-Re(Arg¹¹)CCMSH (13), whereas the melanoma uptake values were19.39±1.65 and 17.01±2.54% ID/g at 2 and 4 h post-injection for¹¹¹In-DOTA-Nle-CycMSH_(hex) (19). Meanwhile, ¹¹¹In-DOTA-Nle-CycMSH_(hex)showed similar tumor/kidney uptake ratios as ¹¹¹In-DOTA-Re(Arg¹¹)CCMSHat 2 and 24 h post-injection (19). In this study, the introduction ofthe -GlyGly-linker maintained high melanoma uptakes of¹¹¹In-DOTA-GGNle-CycMSH_(hex) (19.05±5.04 and 18.6±3.56% ID/g at 2 and 4h post-injection, respectively) compared to ¹¹¹In-DOTA-Nle-CycMSH_(hex).Interestingly, the introduction of -GlyGly-linker reduced the liver andrenal uptakes of ¹¹¹In-DOTA-GGNle-CycMSH_(hex).¹¹¹In-DOTA-GGNle-CycMSH_(hex) exhibited 61, 65 and 68% less liver uptakevalues than ¹¹¹In-DOTA-Nle-CycMSH_(hex) (FIG. 11), and 28, 32 and 42%less renal uptake values than ¹¹¹In-DOTA-Nle-CycMSH_(hex) at 2, 4 and 24h post-injection (FIG. 11), respectively. The maintained high melanomauptakes coupled with the decreased liver and renal uptakes resulted inenhanced tumor liver and tumor/kidney uptake ratios for¹¹¹In-DOTA-GGNle-CycMSH_(hex) compared to ¹¹¹In-DOTA-Nle-CycMSH_(hex) at2 and 4 h post-injection (FIG. 12). The tumor/liver uptake ratios of¹¹¹In-DOTA-GGNle-CycMSH_(hex) were 2.52 and 3.13 times the tumor/liveruptake ratios of ¹¹¹In-DOTA-Nle-CycMSH_(hex) at 2 and 4 hpost-injection, whereas the tumor/kidney uptake ratios of¹¹¹In-DOTA-GGNle-CycMSH_(hex) were 1.37 and 1.61 times the tumor/kidneyuptake ratios of ¹¹¹In-DOTA-Nle-CycMSH_(hex) at 2 and 4 hpost-injection.

As showed in FIG. 9, the enhanced tumor/liver and tumor/kidney uptakeratios of ¹¹¹In-DOTA-GGNle-CycMSH_(hex) generated high tumor imagingcontrast to the background. The flank melanoma lesions were clearlyvisualized by SPECT/CT using ¹¹¹In-DOTA-GGNle-CycMSH_(hex) as an imagingprobe, highlighting its potential as an effective imaging agent formelanoma detection. Furthermore, from the therapeutic point of view, theenhanced tumor/liver and tumor/kidney uptake ratios of¹¹¹In-DOTA-GGNle-CycMSH_(hex) would decrease the absorbed doses to theliver and kidneys when using the therapeutic radionuclide-labeledDOTA-GGNle-CycMSH_(hex) for melanoma treatment. In other words, theimprovement of tumor/liver and tumor/kidney uptake ratios wouldpotentially increase the absorbed dose to the tumor while keeping theliver and kidneys safe when treating the melanoma with the therapeuticradionuclide-labeled DOTA-GGNle-CycMSH_(hex).

TABLE 1A DOTA-conjugated lactam bridge-cyclized alpha-MSH peptides.^(a)DOTA-Nle- DOTA-GGNle- DOTA-GENle- DOTA-NleGE- CycMSH_(hex)CycMSH_(hex) CycMSH_(hex) CycMSH_(hex) Amino acid linker -Nle- -Gly-Gly--Gly-Glu-Nle- -Nle-Gly-Glu- between DOTA and the Nle- cyclic peptidemoiety Calculated molecular 1368.5 1482.6 1554.6 1554.6 weight (Da)Found molecular weight 1368.2 1482.0 1554.0 1554.0 (Da) MolecularFormula C₆₄H₉₃N₁₉O₁₅ C₆₈H₉₉N₂₁O₁₇ C₇₁H₁₀₃N₂₁O₁₉ C₇₁H₁₀₃N₂₁O₁₉ MC1Rbinding affinity 1.8 2.1 11.5 873.4 (nM) HPLC retention time 14.3 14.815.4 9.6 (min) HPLC retention time for 10.7 17.7 21 .7 N/A¹¹¹In-conjugate (min) ^(a)The Data of DOTA-Nle-CycMSH_(hex) was citedfrom Reference 19 for comparison.

Conclusions

The amino acid linkers exhibited profound effects on the melanomatargeting and pharmacokinetic properties of the ¹¹¹In-labeled lactambridge-cyclized α-MSH peptides. Introduction of the -GlyGly-linkermaintained high melanoma uptake while reducing the renal and liveruptakes of ¹¹¹In-DOTA-GlyGlyNle-CycMSH_(hex), highlighting its potentialas an effective imaging probe for melanoma detection, as well as atherapeutic peptide for melanoma treatment when labeled with atherapeutic radionuclide.

The terms and expressions that have been employed in this applicationare used as terms of description and not of limitation, and there is nointent in the use of such terms and expressions to exclude anyequivalent of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention as claimed. Thus, it will be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize that,the invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

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REFERENCES (SECOND SET FOR FURTHER EXAMPLES SECTION)

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1. A compound according to the chemical structure:(Y¹)_(q)-X_(m)-(ABC)_(n)-CycMSH_(hex) Where Y¹ is a chelate group,wherein Y¹ optionally incorporates or complexes with a radioisotope;Each X is independently an amino acid residue which may be optionallyacylated at its amino terminal end or an amino acid linker according tothe chemical structure:

ABC is an amino acid linker wherein A is absent or is a neutral ornegatively charged amino acid at physiological pH which is optionallyacylated at its amino terminal end; B is a neutral or negatively chargedamino acid at physiological pH which is optionally acylated (preferablyC₂-C₂₀ acylated) at its amino terminal end; C is absent or is a neutralor negatively charged amino acid at physiological pH; m is an integerfrom 0 to 250, preferably 0 to 5, preferably 0 or 1; n is 0 or 1,preferably 1; p is an integer from 0 to 20, preferably 0 to 10; k is aninteger from 0 to 10, preferably 1 or 2; s is an integer from 0 to 10,preferably 0, 1 or 2; i is an integer from 0 to 10, preferably 1 or 2; qis 0 or 1, and CycMSH_(hex) is a cyclic peptide comprising six aminoacids according to the general structure:

Wherein W is a C—H group from an aspartic acid or glutamic acid residue(preferably an aspartic acid residue), wherein the alkylene carboxylicacid sidechain of said aspartic acid or glutamic acid and thealkyleneamine sidechain of lysine are bonded together to form an amidelinkage as indicated; X¹ is phenylalanine, tyrosine or tryptophan,preferably D-phenylalanine; Y is arginine or lysine, preferablyarginine; Z is tryptophan, phenylalanine or tyrosine, preferablytryptophan; Z′ is Lys(CONH₂) or Orn(CONH₂), preferably Lys(CONH₂); j is1 or 2 (preferably 1) or a pharmaceutically acceptable salt thereof,wherein said compound is optionally complexed with at least oneradioisotope, wherein said radioisotope is polycationic.
 2. The compoundaccording to claim 1 wherein n is
 0. 3. The compound according to claim1, wherein j is
 1. 4. The compound according to claim 1 wherein q is 1,m is 0, n is 1, j is 1 and X¹ is D-phenylalanine.
 5. The compoundaccording to claim 1 wherein Y is arginine.
 6. The compound according toclaim 1 wherein Z is tryptophan and Z′ is Lys(CONH₂).
 7. The compoundaccording to claim 1 wherein Y¹ is a radical of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane(CB-TE2A), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),Diethylenetriaminopentaacetic acid (DTPA), Mercaptoacetyltriglycine(MAG₃) or 4,5-bis(2-mercaptoacetamido)pentanoic acid or HYNIC(hydrazinonicotinamide).
 8. The compound according to claim 1 wherein Y¹is a radical of DOTA, NOTA or HYNIC.
 9. The compound according to claim1 wherein Y¹ is DOTA.
 10. The compound according to claim 1 wherein j is1, X is norleucine, X¹ is D-phenylalanine, Y is arginine, Z istryptophan, in is 1 and n is
 0. 11. The compound according to claim 1wherein m is 0 and n is
 1. 12. The compound according to claim 1 whereinm is 1 and n is
 1. 13. The compound according to claim 1 wherein n is 1,A is absent, glycine, glutamic acid, aspartic acid or norleucine, B isglutamic acid, aspartic acid, glycine or norleucine and C is absent,norleucine, glutamic acid or aspartic acid.
 14. The compound accordingto claim 13 wherein X is Nle, Gly or a

group, where s is 0 or 1, i is 0 or 1 and k is 1 or
 2. 15. The compoundaccording to claim 1 wherein q is 1, X¹ is D-phenylalanine, Y isarginine, and Z is tryptophan.
 16. The compound according to claim 15wherein j is
 1. 17. The compound according to claim 15 wherein Y¹ is aradical of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane(CB-TE2A), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),Diethylenetriaminopentaacetic acid (DTPA), Mercaptoacetyltriglycine(MAG₃), 4,5-bis(2-mercaptoacetamido)pentanoic acid or HYNIC. 18-39.(canceled)
 40. A pharmaceutical composition comprising an effectiveamount of a compound comprising a radioisotope according to claim 1 incombination with a pharmaceutically acceptable carrier, additive orexcipient. 41-50. (canceled)
 51. A method of treating melanoma in apatient in need of therapy comprising administration to said patient aneffective amount a composition according to claim
 40. 52. (canceled) 53.A method of monitoring therapy of a patient in the treatment ofmelanoma, said method comprising administering to a patient undergoingmelanoma treatment an imaging effective amount of a compound accordingto claim 1, imaging said patient to determine if tissue in said patientexhibits elevated expression of MSH receptors; and comparing the resultsof said imaging step with a standard. 54-63. (canceled)